title: GSM6942879: HCC_Non-Tumor_scRNAseq; Homo sapiens; RNA-Seq
library_name: GSM6942879
library_construction_protocol: Non-tumor and Tumor HCC liver tissues were gently dissociated by mechanical dissection to using forceps and scissors in Collagenase/Dispase (Roche 10269638001) over 2-3 hours at 37°C. Cells were then passed through a 100µm cell strainer and washed with 30ml Advanced DMEM/F12 (Gibco 12634-010) and centrifuged at 100g for 5 mins at 4°C. Red blood cells were lysed by resuspending cell pellet in RBC lysis buffer (Invitrogen 00-4333-57) for 1 minute, diluted with 30ml cold Advanced DMEM/F12 and centrifuged again. Cells were briefly washed cold Advanced DMEM/F12 containing 0.5U/ml RQ1 RNase-free DNaseI (Promega M6101), diluted with 30ml cold Advanced DMEM/F12 and centrifuged again. The live cells were resuspended in cold Advanced DMEM/F12, 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 human HCC scRNA sequencing experiments, 2 generated libraries were pooled and sequenced in one lane using HiSeq X for 367M total Paired end reads.