This method can have answer key questions in the pancreatic islet field about pancreatic islet lineage differentiation, maturation, and the regeneration. The main advantage of this technique is that it allows the collection of single pancreatic islet cells for the generation of high quality single cell RNA sequencing data. The application of this technique extend toward the therapy of diabetes and the other pancreatic endocrine related diseases through the transcriptomic analysis of pancreatic endocrine single cells on the pathological conditions.
Visual demonstration of this method is critical as the insertion of the needle into the narrow bile duct of the pancreas for the perfusion can be tricky. For embryonic day 17.5 embryos. After opening the abdominal cavity, use elbow tweezers to transfer the visceral tissue to a six centimeter black bottom dish containing cold PBS.
Use forceps to detach the pancreas from the visceral tissue. And place the pancreatic tissue in a 20 milliliter vial containing five milliliters of cold collagenase solution. For post natal day zero to 15 mice, carefully dissect all of the lobes of the pancreas before placing the tissue into the collagenase.
For post natal day 18 to 60 mice, first remove the xiphoid and extract the bowel to the right side of the abdominal cavity. Push the left and right medial lobes of the liver to each side to expose the gall bladder and the common bile duct. And clamp the duodenum with a pair of small vessel clamps at the upper and lower positions to flank the site where the bile duct enters the duodenum.
Next, load a 5 milliliter syringe equipped with a 30 gauge needle with cold collagenase solution. And use the microscope to insert the needle tip into the gall bladder. Carefully and smoothly manipulate the needle through the common bile duct.
And depress the plunger to perfuse the pancreas with one to five milliliters of the collagenase solution according to the size of the mouse. When all of the solution has been perfused, use forceps to immediately transfer the pancreas to a 20 milliliter vial containing five milliliters of fresh cold collagenase solution. When all of the pancreatic tissue has been collected, place the vial in a 37 degree Celsius water bath for three minutes, followed by three to five minutes of gentle shaking.
Then filter the digested product through a 0.25 millimeter nylon strainer into a new 50 milliliter centrifuge tube. And use a 20 milliliter syringe containing ice cold PBS to thoroughly wash the strainer. For post natal day 18 to 60 pancreata, centrifuge the filtered sample and resuspend the tissue in 30 milliliters of cold PBS.
Then decant the tissue suspension into a six centimeter black bottom dish, and use a 200 microliter pipette and a dissecting microscope to pick the islets for transfer into a 1.5 milliliter microcentrifuge tube containing a small volume of cold PBS. For embryonic day 17.5 post natal day 15 pancreata, centrifuge the filtered pancreatic tissue and resuspend the pellet in trypsin-EDTA solution for a four minute incubation in a 37 degree Celsius water bath. At the end of the incubation, gently try to rate the pellet for one minute with a 200 microliter pipette, followed by the addition of 0.5 times the volume of cold fetal bovine serum with a gentle vortexing to stop the digestion.
Then collect the digest by centrifugation and resuspend the cells in 200 microliters of FACS buffer in a 5 milliliter FACS tube for sorting by flow cytometry. For single cell picking and lysis, aliquot freshly prepared cell lysis buffer into eight strip PCR tubes, and transfer a single cell into each. Vortex the samples to lyse the cells for release of the RNA, followed by centrifugation for 30 seconds at four degrees Celsius, and immediate storage on ice.
For reverse transcription, incubate the lysates for three minutes at 72 degree Celsius followed by one minute on ice. Briefly centrifuge the samples for 30 seconds at four degree Celsius and add 5.7 microliters of reverse transcription mix to each sample to bring the total volume to 10 microliters for each sample. After gentle vortexing and centrifugation, load the samples into a thermocycler and start the reverse transcription program.
For polymerase chain reaction preamplification, or PCR, add 15 microliters of PCR mix to each sample containing the first strand reactions, followed by gentle vortexing and centrifugation. Then load the samples into the thermocycler and start the PCR program. For PCR purification, add 25 microliters of resuspended DNA purification beads to each sample, and mix well by vortexing.
Then quickly spin the samples at room temperature to collect the liquid while avoiding settlement of the beads. After five minutes at room temperature, place the samples on an appropriate magnetic stand, carefully removing and discarding the supernatant when the solution is clear. Wash the beads with two 30 second washes in 200 microliters of freshly prepared 80%ethanol per wash.
And discard the remaining ethanol after the second wash. When the beads have air dried, add 11 microliters of nuclease-free water to elute the DNA target from the beads, followed by vortexing. Then quickly centrifuge the samples, return the samples to the magnetic stand until the solution is clear and transfer 10 microliters of each sample to a new PCR tube.
Successfully amplified cDNA should have a full length above 500 base pairs, and be enriched from 1.5 to two kilo base pairs. Moreover, there is usually a 500 to 600 base pair enrichment of cDNA observed in insulin one RFP plus cells, which may represent the insulin transcripts. However, the cDNA fragments near 100 base pairs are primer dimers, which are usually caused by excessive primers and which must be removed by repeating the DNA purification step.
The cDNA fragments between 100 and 500 base pairs usually represent degraded cDNA, which is caused by bad cell status or region problems, such as RNase contamination and should be excluded. After the purification of the cDNA libraries for sequencing, cDNA ranging from 250 to 450 base pairs can be obtained through the addition of DNA purification beads to the first and second rounds of purification. After DNA bead purification, the sequencing quality score should be greater than 30 during the sequencing quality evaluation.
To obtain high quality cells for downstream analyses, cells with fewer than 0.5 million mapped reads or with fewer than 4, 000 detected genes are excluded, facilitating the characterization of different groups of cells and the identification of genes that are heterogeneously expressed in different groups after principal component analysis and hierarchical clustering. While attempting this procedure, it's important to remember to perfuse the pancreas quickly before the collagenase digestion to avoid islet over digestion and to maintain RNase free environment during the single cell procedures. Following this procedure, other methods like islet imaging and culture can be performed to facilitate visualization of the islet structure and to examine cellular responses and drug stimulation.
After development, this technique paved the way for researchers in the field of pancreatic research to explore the mechanisms of islet lineage development and regeneration, as well as diabetes progression in mammals.
