GSM8551860: MDOI; Mus musculus; RNA-Seq
mPFC of C57BL/J mice were microdissected and frozen on dry ice. Nuclei isolation ws performed using  Chromium Nuclei Isolation with RNase inhibitor Kit (10x Genomics) according to manufacturer's instructions. Briefly, tissue was dissociated with pestle in lysis buffer and incubated on ice in lysis buffer. Dissociated tissue was pipetted into Nuclei Isolation Column and Collection tube and vortexed to resuspend. Supernantant was removed and resultant pellet was resuspended in Debris Removal buffer and vortexed. Supernatant was removed, washed in Resuspension buffer, and vortexed. Nuclei concentration was determined. Library was constructed using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics) according to manufacturer's instructions. Nuclei and partitioning oil are combined to create Master Mix which is combined with barcoded Gel Beads onto Chromium Next GEM Chip M. The Gel Bead is then dissolved, primers are released, and any copartitioned cell is lysed. Primers containing an Illumina TruSeq Read 1, 16nt 10x Barcode, 12 nt unique molecular identifier (UMI), and 30 nt poly(dT) sequence are mixed with both the cell lysate and a Master Mix containing reverse transcription (RT) reagents. Incubation of the GEMs produces barcoded, full-length cDNA from poly-adenylated mRNA. After incubation, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEMRT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5 and P7 adapters, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification.