GSM7091554: Liver_scRNA_Adar1wt/wt; Mus musculus; RNA-Seq
Liver cell isolation was performed via two-step collagenase perfusion on Ifih1-/- Adar1wt/wt Alb-Cre+, Ifih1-/- Adar1wt/flox Alb-Cre+, and Ifih1-/- Adar1flox/flox Alb-Cre+ mice livers. The inferior vena cava of fresh sacrificed mice was punctured with an 18G perfusion needle, and 30ml 37°C warmed perfusion buffer (1x HBSS) was gently perfused through the liver over the course of 5 minutes until the liver appeared pale. Without disturbing the position of the perfusion needle, 30ml 37°C warmed digestion buffer (Collagenase IV in serum-free DMEM) was then perfused through the liver over the course of 5 minutes until the liver became soft. The liver was then aseptically removed from the mouse into cold DMEM containing 10%FBS. After gross dissection of liver lobes, the lobes were perforated using an 18G needle and dissociated hepatocytes were gently released into the media using a cell scraper. Cells were then passed through a 100µm cell strainer and centrifuged at 50g for 5 mins at 4°C. Red blood cells were lysed by resuspending cell pellet in 3ml Ack Lysing buffer (Gibco A10492-01) for 1 minute, diluted with 30ml cold DMEM containing 10%FBS and centrifuged again. live cells were isolated using 10% Percoll (Sigma P1644) gradient centrifugation at 200g for 15 mins at room temperature. The supernatant was aspirated, and remaining cell pellet were briefly washed with cold DMEM containing 10%FBS and 0.5U/ml RQ1 RNase-free DNaseI (Promega M6101), diluted with 30ml cold DMEM containing 10%FBS and centrifuged again. The live cells were resuspended in cold DMEM containing 10%FBS, passed through a 40µm cell strainer to obtain single cell suspensions, and stained with trypan blue cell stain for viability assessment and manual cell counting prior to scRNA sequencing protocol. 10x Genomics Chromium Next GEM Single Cell 3' kit v3.1 (Dual index) (CG000315 Rev A) was used according to manufacturer's protocol. Briefly, cell suspensions were loaded onto Chromium Next GEM Chip G for Gel-in-emulsion (GEM) generation at concentrations targeting 10,000 cells recovery per sample along with barcoded Single Cell 3' V3.1 gel beads for poly-adenylated mRNA capture and partitioning oil. Only cell suspensions with more than 70% viability (determined by trypan blue stain) were used. Following GEM generation, partitioned cells were lysed and captured RNA were reverse transcribed to first-strand cDNA. GEMs were then broken and Dynabeads MyONE SILANE (10x, 2000048) were used for cleanup before full-length, barcoded cDNA was PCR amplified for library construction. Library preparation was performed according to manufacturer's protocol (CG000315 Rev A). This involved enzymatic fragmentation and size selection using SPRIselect Reagent (Beckman Coulter, B23317) to yield libraries containing P5 and P7 Illumina primer sequences, Sample indexes i5 and i7 (10x, 3000431), TruSeq Read 1 and 2 sites, 10x Barcode, Unique Molecular Identifier (UMI), and inserts. For mice liver scRNA sequencing experiments, 3 generated libraries were pooled and sequenced in one lane using HiSeq X for 367M total Paired end reads.