NextSeq 550 paired end sequencing; scRNA-seq in 79 fibroblast cell lines and 31 reprogrammed induced pluripotent stem cell lines for sceQTL analysis
scRNA-seq in 79 fibroblast cell lines and 31 reprogrammed induced pluripotent stem cell lines for sceQTL analysis
Pool 2_p
Human skin punch biopsies were obtained from subjects over the age of 18 years. Fibroblasts were cultured in DMEM high glucose supplemented with 10% foetal bovine serum (FBS), L-glutamine, penicillin (100 U/mL), streptomycin 100 (µg/mL) (all from Thermo Fisher Scientific, USA). All cell lines were mycoplasma-free (MycoAlert mycoplasma detection kit, Lonza, Switzerland).  Human iPSCs were reprogrammed from fibroblast cultures by nucleofection (AmaxaTM NucleofectorTM) of episomal vectors expressing OCT-4, SOX2, KLF4, L-MYC, LIN28 and shRNA against p53 (40), in feeder- and serum-free conditions using TeSRTM-E7TM medium (STEMCELL Technologies, Canada) and selected by sorting with anti-human TRA-1-60 Microbeads using a MultiMACS (Miltenyi, Germany) as described in (41) and (42). Cells were maintained on vitronectin XFTM (STEMCELL Technologies) -coated plates using TeSRTM-E8TM (Stem Cell Technologies). At passage eight, cells were assessed for quality control as described previously (42).  Viable cells were sorted on a BD Influx cell sorter (Becton-Dickinson) using Propidium Iodide into Dulbecco's phosphate buffered saline (PBS) + 0.1% bovine serum albumin and retained on ice. Sorted cells were counted and assessed for viability with Trypan Blue using a Countess automated counter (Invitrogen), and then resuspended at a concentration of 800-1000 cells/µL (8*105 to 1*106 cells/mL). Final cell viability estimates ranged between 92-96%.   Single cell suspensions were loaded onto 10X Genomics Single Cell 3' Chips along with the reverse transcription (RT) mastermix as per the manufacturer's protocol for the Chromium Single Cell 3' Library (10X Genomics; PN-120233), to generate single cell gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: 55oC for 2h; 85oC for 5min; hold 4oC. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; Cat# 37002D) and SPRIselect beads (Beckman Coulter; Cat# B23318). Purified cDNA was amplified as follows: 98oC for 3min; 12x (98oC for 15s, 67oC for 20s, 72oC for 60s); 72oC for 60s; hold 4oC. Amplified cDNA was purified using SPRIselect beads and sheared to approximately 200bp with a Covaris S2 instrument (Covaris) using the manufacturer's recommended parameters. Sequencing libraries were generated with unique sample indices (SI) for each chromium reaction.