GSM5469966: NLGN3 R451C A; Homo sapiens; RNA-Seq
For each cell line, two wells of a six well plate was plated with mixed cultures at 2x106 iNs and 2.5 x 105 glia/well.  On day 40, cells were dissociated with TrypLE (Thermo Fisher) for 5 min at 37°C, which preferentially releases neurons from glia. Cells were washed twice with 1X HBSS (Thermo Fisher), resuspended at 1200 cells/ml in 1% BSA/1X PBS and placed on ice. Single-cell cDNA libraries were generated using the Chromium Next GEM Single Cell 3' GEM Reagent Kits v3.1 (10X Genomics).  Approximately 2x104dissociated cells were loaded onto a cassette in the Chromium Controller with accompanying reagents to generate Gel Beads in Emulsions (GEMs). These were reverse transcribed to single-cell cDNA libraries before the oil emulsion was disrupted and cDNA purified using Dynabeads MyOne Silane (Thermo Fisher). cDNA was amplified by PCR for 11 cycles and purified using SPRIselect reagent (Beckman-Coulter).  cDNA fragmentation, A-tailing, and end repair was performed followed by adapter ligation for paired-end sequencing.  An additional 12 cycles of PCR were performed to incorporate the sample index sequences and to amplify the libraries before purification and sequencing. scRNAseq