NextSeq 500 sequencing; Single cell RNA-seq of WTC-CRISPRi human induced pluripotent stem cells
Single cell RNA-seq of WTC-CRISPRi human induced pluripotent stem cells
For scRNA-seq, samples one and two were harvested from a single plate using Versene, split into two technical replicates resuspended in Dulbecco's PBS (dPBS; Life Technologies, cat. no. 14190-144) with 0.04% bovine serum albumin (Sigma; , cat. no. A9418-50G) and immediately transported for cell sorting. For samples 3-5 cells were harvested from individual plates using 0.25% Trypsin (Life Technologies, cat. no. 15090-046) in Versene, neutralized using 50% fetal bovine serum (HyClone, cat. no. SH30396.03) in DMEM/F12 (Life Technologies, cat. no. 11320-033), centrifuged at 1200 rpm for 5 minutes and re-suspended in dPBS + 0.04% BSA.Viable cells were sorted on a BD Influx cell sorter (Becton-Dickinson) using Propidium Iodide into Dulbecco's PBS + 0.04% 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 x 105-1 x 106 cells/mL). Final cell viability estimates ranged between 80-93%. Undifferentiated human induced pluripotent stem cells (hiPSC; WTC-wild type C) were provided courtesy of Bruce Conklin (UCSF and Gladstone Institutes) as previously described (Mandegar et al., 2016). Cells were maintained on Vitronectin XF (STEMCELL Technologies, cat. no. 07180) and cultured in mTeSR1 (STEMCELL Technologies, cat. no. 05850). Cytogenetic analysis by Giemsa banding showed a normal 46, XY male karyotype.  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; version 1 chemistry; 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 sample.