Name: chromatin isolation by rna purification (chirp)
Uploaded: 2012-03-25T14:00:00
Duration: 11 min 9 s
Description: Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

We thank T. Hung, MC. Tsai, O. Manor, E. Segal, M. Kuroda, T. Swigut, and I. Shestopalov for discussions. Supported by the Agency of Science, Technology and Research of Singapore (C.C.), NIH R01-CA118750 and R01- HG004361 (H.Y.C.), and California Institute for Regenerative Medicine (H.Y.C.). H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute.

1. Probe Design
Design anti-sense DNA tiling probes for selective retrieval of RNA target by ChIRP.
<ol>
	<li>Design anti-sense oligo probes using the online probe designer at <a href="http://singlemoleculefish.com">singlemoleculefish.com</a> 18.</li>
	<li>Use these parameters: number of probes = 1 probe /100 bp of RNA length; 2) Target GC% = 45; 3) Oligonucleotide length = 20; 4) Spacing length = 60-80. Break RNA into segments if too long for the designer. Omit regions of repeats or extensive homology.</li>
	<li>Order anti-sense DNA probes with BiotinTEG at 3-prime end.</li>
	<li>Label probes according to their positions along the RNA. Separate them into two pools so that the "even" pool contains all probes numbering 2, 4, 6, etc. and the "odd" pool contains probes numbering 1, 3, 5, etc. Dilute pool of probes to 100 &mu;M concentration and store at -20 &deg;C.</li>
	<li>All experiments are to be performed using both pools, which serve as internal controls for each other. Real RNA-dependent signal would be present from both pools, while probe-specific noises would be unique to each pool. This applies for both ChIRP-qPCR and ChIRP-seq.</li>
</ol>
2. Harvest Cells
Collect cells that will be used for ChIRP experiment.
<ol>
	<li>Grow cells in tissue culture plates or flasks to confluency. Rinse with phosphate buffered saline (PBS) once and trypsinize. Quench trypsin with &gt;2x volume of media, pipette up and down to dislodge cells and resuspend into single cell suspension. Transfer all media and resuspended cells into 50 ml Falcon tubes. 20 million cells are typically sufficient for one ChIRP sample.</li>
	<li>Spin cells at 800RCF for 4 min. Aspirate media and resuspend 40 million cells in 40 ml of PBS, combine tubes if necessary. Spin cells at 800RCF for 4 min. Decant PBS, carefully aspirate on an angle the remaining liquid.</li>
</ol>
3. Cross-link Cells and Collect Cell Pellet
Crosslink collected cells with glutaraldehyde to preserve RNA-Chromatin interactions and prepare cell pellet.
<ol>
	<li>Perform all steps at room temperature.</li>
	<li>Prepare 1% glutaraldehyde in room temperature PBS. Prepare 10 ml per 10 million cells (0.4 mL 25% glutaraldehyde stock + 9.6 mL PBS). Glutaraldehyde must be used fresh.</li>
	<li>Tap bottom of Falcon tubes to dislodge pellets. Resuspend cell pellet in 1% glutaraldehyde, starting with a small volume to avoid chunks, then top up to full volume. Invert to mix. Crosslink for 10min at room temperature on an end-to-end shaker or rotator.</li>
	<li>Quench the cross-linking reaction with 1/10th volume of 1.25 M glycine at room temperature for 5 min.</li>
	<li>Spin at 2000RCF for 5 min. Aspirate supernatant and wash pellet with 20 mL chilled PBS once, spinning at 2000RCF for 5 min.</li>
	<li>Aspirate and resuspend the washed, cross-linked pellet with 1 ml chilled PBS per 20 million cells. Transfer each ml to an Eppendorf tube and spin at 2000RCF for 3 min at 4 C. Remove as much PBS as possible with pipette tip carefully.</li>
	<li>Flash-freeze the cell pellets in liquid nitrogen and store at -80 &deg;C indefinitely.</li>
</ol>
4. Cell Lysis
Lyse crosslinked cells to prepare cell lysate.
<ol>
	<li>Thaw frozen cell pellets at room temperature. Tap hard to dislodge and mix the cell pellet. Spin down the pellet at 2000RCF for 3 min at 4 &deg;C. Use a sharp 10 &mu;l pipette tip to remove any remaining PBS.</li>
	<li>On an electronic balance (accurate to 1mg) tare the mass of an empty Eppendorf tube (our tubes weigh 1.060 grams very consistently). Weigh each pellet and record its weight. A full 15 cm dish of crosslinked HeLa cells typically weighs 100 mg.</li>
	<li>Supplement Lysis Buffer (10X the mass of pellet, e.g. 1 ml for 100mg) with fresh Protease Inhibitor, PMSF and Superase-in (see attached buffer list). Mix well.</li>
	<li>Add 10X volume supplemented Lysis Buffer to each tube and resuspend the pellet. For small pellets &lt;25 mg, resuspend in 250 &mu;l supplemented lysis buffer. Suspension should be smooth. If not, divide suspension into 500 &mu;l aliquots and use a motorized pellet mixer to break up clumps. Proceed immediately to sonication.</li>
</ol>
5. Sonication
Shear DNA by sonicating crosslinked cell lysates.
<ol>
	<li>Sonicate cell lysate in Bioruptor in 15 ml Falcon tubes. Use &lt;1.5 ml lysate in each tube, and for faster sonication, sonicate no more than two tubes at a time.</li>
	<li>Sonicate in a 4 &deg;C water bath at highest setting with 30 seconds ON, 45 seconds OFF pulse intervals. Check lysate every 30 min. Continue sonicating until the cell lysate is no longer turbid. This may take as little as 30 min and as many as 4 hrs. The number of tubes, the sample volume, the bath temperature, and the period of sonication time will affect how long the process takes. Tubes will likely sonicate at different rates, so pool them together every 30 min and redistribute into original tubes to ensure homogeneity. Note: glutaraldehyde-crosslinked cells take significantly longer to sonicate than formaldehyde equivalents.</li>
	<li>When lysate turns clear, transfer 5 &mu;L lysate to a fresh Eppendorf tube. Add 90 &mu;L DNA Protease K (PK) Buffer (see buffer list) and 5 &mu;L PK. Vortex to mix and spin down briefly. Incubate for 45 min at 50 &deg;C.</li>
	<li>Extract DNA with Qiagen PCR purification kit. Elute DNA in 30 &mu;L Qiagen Elution Buffer (EB) and check DNA size on 1% agarose gel. If bulk of the DNA smear is 100-500 bp, sonication is complete. If not, continue to sonicate.</li>
	<li>Centrifuge sonicated samples at 16100RCF for 10 min at 4 &deg;C. Combine supernatants, aliquot into 1 mL samples and flash-freeze in liquid nitrogen. Store at -80 &deg;C.</li>
</ol>
6. ChIRP
Hybridize biotinylated DNA probes to RNA and isolate bound chromatin.
<ol>
	<li>Thaw tubes of chromatin at room temperature.</li>
	<li>Prepare Hybridization Buffer (see buffer list, prepare 2 ml per ml of chromatin). Vortex to mix.</li>
	<li>For a typical ChIRP sample using 1 ml of lysate, remove 10 &mu;L for RNA INPUT and 10 &mu;L for DNA INPUT and place in Eppendorf tubes. Keep on ice till further use.</li>
	<li>Transfer 1 mL chromatin to 15 mL Falcon tube. Add 2 mL Hybridization Buffer to each tube. For total volume &lt;1.5 ml, use Eppendorf tubes.</li>
	<li>Thaw probes at room temperature. Nanodrop probes to check amount if you haven't used it in a long time (100 &mu;M probes should spec ~500-600 ng/&mu;l using single strand DNA setting). Add appropriate volume of probes to specific tubes (100 pmol probe per 1 mL chromatin, 1 &mu;L of 100 pmol/&mu;L probe per 1 mL chromatin). Mix well. Incubate at 37 &deg;C for 4 hrs with shaking.</li>
	<li>With 20 min remaining for hybridization, prepare the C-1 magnetic beads (stored at 4 &deg;C). Use 100 &mu;L per 100 pmol of probes. Wash with 1 mL unsupplemented Lysis Buffer three times, using the DynaMag-2 magnet strip to separate beads from buffer.</li>
	<li>Resuspend beads in original volume of Lysis Buffer; supplement with fresh PMSF, P.I and Superase-in. After 4 hr hybridization reaction is complete, add 100 &mu;L beads to each tube. Mix well. Incubate at 37 &deg;C for 30 min with shaking.</li>
	<li>Prepare Wash Buffer (5 mL per sample). Vortex to mix. Pre-warm to 37 &deg;C. Add PMSF before use.</li>
	<li>Wash beads with 1 mL wash buffer five times. On the first wash, use DynaMag-15 magnetic strip to separate beads, decant, and resuspend in 1 mL wash buffer. Transfer volume to 1.5 mL Eppendorf tube. Incubate at 37 &deg;C with shaking for 5 min.</li>
	<li>On subsequent washes, spin down each tube on a minicentrifuge, set sample on DynaMag-2 magnetic strip for 1 min. Decant sample, wipe any drips with a Kimwipe, resuspend in 1 mL wash buffer. Incubate at 37 &deg;C with shaking for 5 min. Repeat for five total washes.</li>
	<li>At last wash, resuspend the beads well. Remove 100 &mu;L and set aside for RNA isolation. Reserve 900 &mu;L for DNA fraction. Place all tubes on DynaMag-2 magnetic strip and remove wash buffer. Spin all tubes down briefly; place them on magnet strip. Remove the last bit of wash buffer completely with a sharp 10 &mu;l pipette tip.</li>
</ol>
7. RNA Isolation
Extract RNA fraction from ChIRP samples to quantitate by qRT-PCR.
<ol>
	<li>Take 100 &mu;L bead samples and 10 &mu;L RNA INPUT sample. Add 85 &mu;L RNA PK Buffer pH 7.0 to RNA INPUT. Resuspend beads in 95 &mu;L RNA PK Buffer pH 7.0. Add 5 &mu;L Proteinease K and incubate at 50 &deg;C for 45 min with end-to-end shaking.</li>
	<li>Briefly spin down all tubes and boil samples for 10 min on heat block at 95 &deg;C.</li>
	<li>Chill samples on ice, add 500 &mu;L TRIzol, vortex vigorously for 10 sec. Incubate at room temperature for 10 min. Store at -80 &deg;C or proceed to step 4.</li>
	<li>Add 100 &mu;L chloroform to TRIzol treated samples. Vortex vigorously for 10 sec. Spin at 16100RCF on a benchtop centrifuge for 15 min at 4 &deg;C.</li>
	<li>Remove ~400 &mu;L aqueous supernatant, avoiding organic and interface.</li>
	<li>Add 600 &mu;L (1.5 volume) 100% ethanol and mix well. Spin sample through MIRNeasy mini columns. Wash 1x with RWT (MIRNeasy mini kit), 2x with RPE per manufacturer's protocol. Elute with 30 &mu;L nuclease-free H2O (nfH2O).</li>
	<li>Treat the RNA eluate with DNA-free per manufacturer's protocol. After the reaction is complete, heat the sample for 15 minutes at 65 &deg;C to completely inactivate any remaining DNase.</li>
	<li>Use 1 &mu;L RNA isolate per well for qRT-PCR analysis to confirm lncRNA retrieval. GAPDH is often used as a negative control.</li>
</ol>
8. DNA Isolation
Extract DNA fraction from ChIRP samples to identify by sequencing or quantitate by qPCR.
<ol>
	<li>Prepare DNA Elution Buffer (see buffer list), 150 &mu;l per sample, including DNA INPUT.</li>
	<li>Add 1 0&mu;L RNase A (10 mg/mL) and 10 &mu;L RNase H (10 U/&mu;l) per ml of DNA Elution Buffer, and vortex to mix.</li>
	<li>Resuspend each sample of beads in 150 &mu;L of DNA Elution Buffer with RNases. (Resuspend DNA INPUT in 140 &mu;L) Incubate at 37 &deg;C for 30 min with shaking.</li>
	<li>Separate beads and supernatant on DynaMag-2 magnetic strip. Remove supernatant and add to labeled tubes.</li>
	<li>Prepare a second aliquot of DNA Elution Buffer with 10 &mu;L RNase A (10 mg/mL) and RNaseH (10 U/&mu;L) exactly as done in 8.2). Add 150 &mu;L to each sample (including DNA INPUT), incubate, and remove supernatant. Collect all supernatant (should be ~300 &mu;L).</li>
	<li>Add 15 &mu;L PK to each sample. Incubate at 50 &deg;C for 45 min with shaking.</li>
	<li>Pre-spin down yellow phase-lock gel tubes (5PRIME). Transfer DNA samples to phase-lock gel tubes, and add 300 &mu;L PhOH:Chloroform:Isoamyl per sample. Shake vigorously for 10 min, and spin down on a benchtop centrifuge at 16100RCF for 5 min at 4 &deg;C. Take aqueous from the top (~300 &mu;L). Add 3 &mu;L GlycoBlue, 30 &mu;L NaOAc, and 900 &mu;L 100% EtOH. Mix well and store at -20 &deg;C overnight.</li>
	<li>Spin samples at 16100RCF for 30 min at 4 &deg;C.</li>
	<li>Decant supernatant carefully. Add 1 mL 70% EtOH and vortex to mix. Spin at 16100RCF for 5 min. Remove supernatant by pipette. Air dry for 1min. Resuspend in 30 &mu;L EB.</li>
	<li>DNA samples are ready for analysis by qPCR or preparation of high-throughput sequencing libraries per Illumina protocol.</li>
</ol>
10. Representative Results
Figure 1 depicts the ChIRP workflow. A successful ChIRP experiment typically enriches target RNA significantly over non-specific RNAs. Figure 2 shows enrichment of human telomerase RNA (TERC) from HeLa cells over GAPDH, an abundant cellular RNA that serves as a negative control. Majority of TERC RNAs (~88%) present in the cell were pulled down by performing ChIRP, whereas only 0.46% of GAPDH RNA was retrieved, demonstrating an enrichment factor of ~200 fold. Nonspecific probes such as probes targeting LacZ RNA, which is not expressed in mammalian cells (Figure 2), can be used as additional negative controls.
DNA regions expected to bind the target lncRNA are typically enriched over negative regions when measured by qPCR. Figure 3 shows qPCR validation of four HOTAIR-bound sites in primary human foreskin fibroblasts that we determined by performing ChIRP-seq in the same cell line, while TERC and GAPDH DNA sites serve as negative control regions. Both "even" and "odd" probe sets yielded comparable enrichment of expected HOTAIR-bound sites over negative regions, a hallmark of true lncRNA-binding sites.
High-throughput sequencing of ChIRP enriched DNA yields a global map of lncRNA-binding sites. The Drosophila lncRNA roX2 is known to interact with the X-chromosome in a manner that is required for dosage compensation. Figure 4 shows roX2 binding profile over a section of the X chromosome. Both "even" and "odd" samples have been sequenced and their unique noises have been eliminated to produce a track of overlapping signals. Each "peak" here indicates a strong site of roX2 binding. The complete track and list of roX2 target genes have been described in Chu et al. 2011 17.
<img alt="Figure 1." src="/files/ftp_upload/3912/3912fig1.jpg" /> 
Figure 1. Flow chart of the ChIRP procedure. Chromatin is crosslinked to lncRNA:protein adducts in vivo. Biotinylated tiling probes are hybridized to target lncRNA, and chromatin complexes are purified using magnetic streptavidin beads, followed by stringent washes. We elute lncRNA bound DNA or proteins with a cocktail of Rnase A and H. A putative lncRNA binding sequence is schematized in orange. Previously published in Chu et al. 2011.17
<img alt="Figure 2." src="/files/ftp_upload/3912/3912fig2.jpg" /> 
Figure 2. ChIRP enriches for human TERC RNA. TERC-asDNA probes retrieve ~88% of cellular TERC RNA and undetectable GAPDH. LacZ-asDNA probes are used as negative controls and retrieve neither RNAs. Mean + s.d. are shown. Previously published in Chu et al. 2011.17
<img alt="Figure 3." src="/files/ftp_upload/3912/3912fig3.jpg" /> 
Figure 3. HOTAIR ChIRP-qPCR in primary human foreskin fibroblasts. NFKBIA, HOXD3-4, SERINC5 and ABCA2 are regions that interact with HOTAIR. TERC and GAPDH served as negative controls. Mean + s.d. are shown. Previously published in Chu et al. 2011.17
<img alt="Figure 4." src="/files/ftp_upload/3912/3912fig4.jpg" /> 
Figure 4. ChIRP-seq data of roX2 RNA in Sl2 Drosophila cells. "Even" and "odd" were sequenced separately; their data merge to reflect only common peaks in both. The merged track is shown. Previously published in Chu et al. 2011.17

<ol>
	<li>Koziol, M. J., Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+traffic+control+of+chromatin+complexes.">RNA traffic control of chromatin complexes.</a> Curr. Opin. Genet. Dev. 20, 142-148 (2010).</li><li>Mercer, T. R., Dinger, M. E., Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNAs:+insights+into+functions.">Long non-coding RNAs: insights into functions.</a> Nat. Rev. Genet. 10, 155-159 (2009).</li><li>Rinn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+demarcation+of+active+and+silent+chromatin+domains+in+human+HOX+loci+by+noncoding+RNAs.">Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.</a> Cell. 129, 1311-1323 (2007).</li><li>Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+proteins+targeted+by+a+short+repeat+RNA+to+the+mouse+X+chromosome.">Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome.</a> Science. 322, 750-756 (2008).</li><li>Kelley, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+spreading+of+the+Drosophila+dosage+compensation+complex+from+roX+RNA+genes+into+flanking+chromatin.">Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin.</a> Cell. 98, 513-522 (1999).</li><li>Pandey, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kcnq1ot1+antisense+noncoding+RNA+mediates+lineage-specific+transcriptional+silencing+through+chromatin-level+regulation.">Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation.</a> Mol. Cell. 32, 232-246 (2008).</li><li>Wang, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+long+noncoding+RNA+maintains+active+chromatin+to+coordinate+homeotic+gene+expression.">A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression.</a> Nature. 472, 120-124 (2011).</li><li>Khalil, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+human+large+intergenic+noncoding+RNAs+associate+with+chromatin-modifying+complexes+and+affect+gene+expression.">Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 11667-11672 (2009).</li><li>Zhao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+polycomb-associated+RNAs+by+RIP-seq.">Genome-wide identification of polycomb-associated RNAs by RIP-seq.</a> Mol. Cell. 40, 939-953 (2010).</li><li>Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=roX1+RNA+paints+the+X+chromosome+of+male+Drosophila+and+is+regulated+by+the+dosage+compensation+system.">roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system.</a> Cell. 88, 445-457 (1997).</li><li>Franke, A., Baker, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rox1+and+rox2+RNAs+are+essential+components+of+the+compensasome,+which+mediates+dosage+compensation+in+Drosophila.">The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila.</a> Mol. Cell. 4, 117-122 (1999).</li><li>Gupta, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+non-coding+RNA+HOTAIR+reprograms+chromatin+state+to+promote+cancer+metastasis.">Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.</a> Nature. 464, 1071-1076 (2010).</li><li>Tsai, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNA+as+modular+scaffold+of+histone+modification+complexes.">Long noncoding RNA as modular scaffold of histone modification complexes.</a> Science. 329, 689-693 (2010).</li><li>Zappulla, D. C., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+as+a+flexible+scaffold+for+proteins:+yeast+telomerase+and+beyond.">RNA as a flexible scaffold for proteins: yeast telomerase and beyond.</a> Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).</li><li>Nagano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Air+noncoding+RNA+epigenetically+silences+transcription+by+targeting+G9a+to+chromatin.">The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.</a> Science. 322, 1717-1720 (2008).</li><li>Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-range+chromatin+regulatory+interactions+in+vivo.">Long-range chromatin regulatory interactions in vivo.</a> Nature. 32, 623-626 (2002).</li><li>Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Maps+of+Long+Noncoding+RNA+Occupancy+Reveal+Principles+of+RNA-Chromatin+Interactions.">Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions.</a> Mol. Cell. , (2011).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat. Methods. 5, 877-879 (2008).</li></ol>

C. Chu and H.Y. Chang are named as inventors on a patent application based on this method.

Here we described ChIRP-seq, a method of mapping in vivo lncRNA binding sites genome-wide. The key parameters for success are the split pools of tiling oligonucleotide probes and glutaraldehyde crosslinking. The design of affinity-probes is straightforward given the RNA sequence and requires no prior knowledge of the RNA's structure or functional domains. Our success with roX2, TERC, and HOTAIR - three rather different RNAs in two species - suggests that ChIRP-seq is likely generalizable to many lncRNAs. As with all experiments, care and proper controls are required to interpret the results. Different lncRNA may require titration of conditions, and judicious change of conditions, such as selection of different affinity probes or crosslinkers, may highlight different aspects of RNA-chromatin interactions. Like ChIP-seq, not all binding events are necessarily functional, and additional studies are required to ascertain the biological consequences of RNA occupancy on chromatin. Nonetheless, we foresee many interesting applications of this technology for researchers of other chromatin-associated lncRNAs, which now number in the thousands8,9. Just as ChIP-seq has opened the door for genome-wide explorations of DNA-protein interactions, ChIRP-seq studies of the "RNA interactome" may reveal many new avenues of biology.

Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20&deg;C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0 
10 mM EDTA 
1% SDS 
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl 
10 mM TrisCl pH 8.0 (For RNA use pH 7.0) 
1 mM EDTA 
0.5% SDS 
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl 
1% SDS 
50 mM Tris-Cl pH 7.0 
1 mM EDTA 
15% formamide (store in the dark at 4&deg;C) 
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock) 
0.5% SDS 
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3 
1% SDS
Table of specific reagents and equipment:
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (EM grade)</td>
			<td>Sigma Aldrich</td>
			<td>G5882-10x10ml</td>
		</tr>
		<tr>
			<td>Motorized pellet mixer</td>
			<td>VWR</td>
			<td>V8185-904</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>78830</td>
		</tr>
		<tr>
			<td>Superase-in</td>
			<td>Ambion</td>
			<td><a href="http://www.ambion.com/catalog/CatNum.php?AM2696">AM2696</a></td>
		</tr>
		<tr>
			<td>Bioruptor</td>
			<td>Diagenode</td>
			<td>UCD-200</td>
		</tr>
		<tr>
			<td>Falcon tubes (for sonication)</td>
			<td>Corning</td>
			<td>430790</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Ambion</td>
			<td>AM2546</td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28106</td>
		</tr>
		<tr>
			<td>C-1 magnetic beads</td>
			<td>Invitrogen</td>
			<td>65002</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma Aldrich</td>
			<td>P7626-25G</td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Invitrogen</td>
			<td>123-01D</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
		</tr>
		<tr>
			<td>MIRNeasy mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
		</tr>
		<tr>
			<td>Rnase H</td>
			<td>Epicentre</td>
			<td>R0601K</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma Aldrich</td>
			<td>R4875-100MG</td>
		</tr>
		<tr>
			<td>Phase Lock Gel Heavy</td>
			<td>5 PRIME</td>
			<td>2302810</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
		</tr>
		<tr>
			<td>Phenol:chloroform:Isoamyl</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Ricca</td>
			<td>RSOC0020-1C</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9515</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>J.T. Baker</td>
			<td>4057-06</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Invitrogen</td>
			<td>10010-049</td>
		</tr>
		<tr>
			<td>Elution Buffer (EB)</td>
			<td>Qiagen</td>
			<td>19086</td>
		</tr>
		<tr>
			<td>20x SSC</td>
			<td>Invitrogen</td>
			<td>15557-036</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>Invitrogen</td>
			<td>15553-027</td>
		</tr>
		<tr>
			<td>DNA-free</td>
			<td>Ambion</td>
			<td>AM1906</td>
		</tr>
		<tr>
			<td>Buffer kit</td>
			<td>Ambion</td>
			<td>AM9010</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Invitrogen</td>
			<td>15515-026</td>
		</tr>
	</tbody>
</table>

chromatin isolation by rna purification (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Watch this Scientific Journal Video about Chromatin Isolation by RNA Purification ChIRP at JoVE.com

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

chromatin-isolation-by-rna-purification-chirp

Research

JoVE Journal

Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...