GSM8899619: sciatic nerve, replicate 5, scRNAseq; Mus musculus; RNA-Seq
12 males and 10 females for P0, 10 males and 11 females for P7, 10 males and 8 females for P14, 9 males and 6 females for P28, and 5 males and 8 females for P60, to facilitate scRNA-seq library preparation. DRGs at the lumbar level of P14 mice were meticulously dissected from 4 males and 4 females to facilitate snRNA-seq library preparation. The harvested neural tissues were placed in a culture dish containing an appropriate volume of calcium-free and magnesium-free PBS on ice, sectioned into 0.5 mm² pieces, and subsequently dissociated into single cells using a dissociation medium containing 0.35% collagenase IV, 2 mg/ml papain, and 120 Units/ml DNase I, maintained in a 37°C water bath. The enzymatic digestion was terminated using 1×PBS with 10% fetal bovine serum. The resulting cell suspension was passed through a 70-30 μm cell strainer and centrifuged at 300 g for 5 minutes at 4°C. The cell pellet was then resuspended in 100 μl of 1× PBS (0.04% BSA) and supplemented with 1 ml of 10× red blood cell lysis buffer, allowing it to incubate at RT for 2-10 minutes. Subsequent centrifugation at 300 g for 5 minutes at room temperature was performed. The cells were resuspended in 100 μl of a Dead Cell Removal MicroBeads solution, and non-viable cells were eliminated using the Miltenyi® Dead Cell Removal Kit. The final cell suspension was prepared in 50 μl of 1× PBS (0.04% BSA). Overall cell viability was verified via trypan blue exclusion, yielding a viability rate exceeding 85%. Single-cell suspensions were quantified using a hemocytometer or a Countess II Automated Cell Counter, with the cell concentration adjusted to 700-1200 cells/μl. R1 (read 1 primer sequence) are added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) are added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.
GEO
GSM8899619_r1