Only and only binding proteins control multiple biological processes. This FLAG-Biotin CLIP-seq method, hence to globally profile the special and the temporal arrangement of RNAs and RBPs in mammalian cells. This method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and the membrane transfer procedures.
Compared to traditional CLIP-seq is isotope free and it doesn't require protein specific antibody. Begin by plating about 3 million mouse embryonic STEM cells in a 10 centimeter plate and incubating them overnight. On the next day, remove the medium with a vacuum and treat the cells with two milliliters of 0.25%trypsin EDTA for two minutes at room temperature.
Quench the trypsin with four milliliters of fresh MESC medium, and transfer the cells to a 15 milliliter centrifuge tube. Then, centrifuge them at 300 times G for three minutes at four degrees Celsius and remove the supernatant. Suspend the cells in four milliliters of cold PBS and transfer them back to the 10 centimeter plate for cross-linking.
Radiate the cells with a 254 nanometer UV light, then transfer the cross-linked cells to a 15 milliliter tube. Spin down this cells at 300 times G for three minutes at four degrees Celsius, then remove the supernatant and lyse the cells with 500 microliters of Buffer A prepared according to manuscript directions. Transfer the cells to an RNAse-free 1.5 milliliter tube, and incubate them at four degrees Celsius with gentle rotation for 30 to 60 minutes.
Treat the lysate with 30 microliters of DNAse 1 at 37 degrees Celsius for 10 minutes. Then, stop the reaction by adding four microliters of 0.5 molar EDTA. Spin down the insoluble pellet by centrifuging at 12, 000 times G for 20 minutes at four degrees Celsius, and transfer the supernatant to a new pre chilled 1.5 milliliter tube.
Transfer the cell lysate to pre equilibrated FLAG beads, and incubate the sample at four degrees Celsius while rotating for two to four hours. After the incubation, centrifuge the beads for two minutes at 3000 times G and remove the supernatant. Add 500 microliters of Buffer A to the sample, and incubated at 4 degrees Celsius for five minutes, then spin down the beads and remove the supernatant.
Repeat the wash with Buffer A, followed by two washes with pre chilled Buffer B.To elute with a three X FLAG-peptide, prepare the elution solution as described in the text manuscript and spin down the FLAG-beads. Remove the supernatant with a narrow end pipette tip and add 200 microliters of the three X FLAG elution solution to each sample. Incubate the samples for 30 minutes at four degrees Celsius with gentle rotation.
Then spin down the beads for two minutes at 3000 times G.Transfer the supernate to a fresh tube and repeat the elution twice. Save 5%of the eluents for Western blot analysis or silver staining to check the efficiency of FLAG immunoprecipitation. Transfer the pulled eluents to prepared streptavidin beads and rotate the samples gently at four degrees Celsius for one to three hours or overnight.
Then, collect the beads on a magnetic stand and remove the supernatant. Wash the beads twice with 500 microliters of Buffer C with gentle rotation for five minutes per wash. Next, wash the beads twice with 500 microliters of Buffer D, and twice with 500 microliters of ice-cold PNK buffer, collecting the beads on the magnetic stand and removing the supernatant in between washes.
To perform partial RNA digestion, add 100 microliters of MNA solution to each sample and vortex set at 37 degrees Celsius with a thermal mixer for 10 minutes. Collect the beads with a magnetic stand for 30 seconds and remove the supernatant. Then, wash them with PNK-EDTA buffer, Buffer C, and PNK buffer according to manuscript directions.
Collect the beads with the magnetic stand and remove the buffer. Then, add the CIP reaction mix and vortex the beads at 37 degrees Celsius with a thermal mixer for 10 minutes. Quickly wash the beads twice with 500 microliters of ice-cold PNK EDTA buffer, followed by two washes with 500 microliters of ice-cold PNK buffer.
After the washes, add 40 microliters of three prime linker ligation mix to the beads, and vortex them at 16 degrees Celsius with a thermal mixer for three hours or overnight. Efficient ligation of the linker to the allies is critical for this experiment. Check the reaction tube occasionally to make sure that these do not precipitate during ligation.
After the incubation, repeat the washes with PNK EDTA and PNK buffers, add 40 microliters of PNK mixed to the beads, and vortex them at 37 degrees Celsius with the thermal mixer for 10 minutes. Wash the beads with PNK EDTA and PNK buffers, then proceed with RNA isolation as described in the text manuscript. Compared with FLAG-mediated or streptavidin mediated one step affinity purification, FLAG-Biotin tandem purification removed almost all the core purified proteins, avoiding the contamination of indirect protein RNA interactions.
LIN28 and WDR43, Fbio CLIP-seq was performed using mouse embryonic STEM cells. In total, about 7.7 million and 2.2 million unique reads were retrieved for LIN28 and WDR43 respectively. Comparison of the track views show different binding patterns in the RN45 pre-rRNA locus.
The cross-linked sites on the GGA G motif of pre-let7-g RNA by LIN28, Fbio CLIP-seq or identified. Furthermore, enriched RNA motifs in the binding sites, and the percentage of mutated nucleotides in different types of mutations were determined. Showing that LIN28 prefers to bind to the gene nucleotide.
Well, at 10:00 PM, this protocol, it is important to confirm the efficiency of the immunoprecipitation to make sure that the protein only complexes were purified successfully.
