GSM7230456
Cell cultures were dissociated into single cells suspension using Papain Dissociation System (Worthington #LK003150), passed through 70μm and 40μm cell strainers (Falcon #07-201-431 and #07-201-430), and resuspended in HIB++ media (HibernateTM-E Medium medium (Gibco #A1247601) supplemented with EDTA (1mM final; Invitrogen #AM9260G), HEPES (10mM final; Gibco #15630080), with 1X B27+ supplement (Gibco #17504-044), 1X N2 supplement (Gibco #17502-048); 1X GlutaMAX(Gibco #35050-061), BDNF (PeproTech #450-02), GDNF (Alomone labs #G-240), CNTF (Alomone labs #C-240), NT-3 (PeproTech #450-03) and IGF-1 (Stem Cell #78022) all at 20 ng/ml)) to 1000 cells per μl using CASY Cell Counter (Innovatis AG) For the single-cell RNA-seq data, 10,000 cells per sample were loaded in to the 10X Chromium controller and library preparation was performed according to the manufacturer's indications (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 protocol). For the bulk RNA-seq data, the quality of the isolated RNA was determined with a Fragment Analyzer (Agilent, Santa Clara, California, USA). The TruSeq Stranded mRNA (Illumina, Inc, California, USA) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were poly A enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR.  The quality and quantity of the enriched libraries were validated using the Fragment Analyzer (Agilent, Santa Clara, California, USA).  The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. For the single-cell RNA-seq data, libraries followed the 10X Genomics recommendations: paired-end reads, R1=28, i7=8, R2=91. For the bulk RNA-seq data, the Novaseq 6000 (Illumina, Inc, California, USA) was used for cluster generation and sequencing according to standard protocol.  Sequencing configuration was paired end 150 bp, 150 million reads depth.