GSM7986403: Transcriptome of late TS543 culture passage; Homo sapiens; RNA-Seq
For PW073 to PW079: At the end of culture, neurospheres were collected and dissociated to single cells by pipetting and stained with with TotalSeq™-A hashtag antibodies (BioLegend), then pooled for scRNA-seq. For PLZ019: Mice were sacrificed for collecting brain tumor tissues 21 post-injection. Freshly resected tumor tissues were kept in ice-cold PBS for transportation prior to dissociation. Mouse brain tumor resections were collected for dissociation using Papain Dissociation System (Worthington) as previously described(Mizrak et al., 2020; Mizrak et al., 2019) with following modifications. Briefly, minced tissue was digested with papain (Worthington, 10 units per sample) in PIPES solution (120 mM NaCl, 5 mM KCl, 20 mM PIPES (Sigma), 0.45% glucose, DNase I (Worthington, 100 units per sample), 1xAntibiotic/Antimycotic (GIBCO), pH adjusted to 7.6) for 30 min at 37°C on gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec, program 37C_ABDK). After digestion, the cell suspension was applied to a MACS SmartStrainer (70 μm, Miltenyi Biotec) and centrifuged at 300xg for 10 minutes at 4°C. The cell pellets were then subjected to red blood cell removal (Red Blood Cell Lysis Solution (10x), Miltenyi Biotec) and debris removal (Debris Removal Solution, Miltenyi Biotec) according to the manufacturer's instructions. For PW073 to PW079: mRNA and hashtag molecues were captured using bead-bound oligo dT(30). Captured mRNA and hashtag molecues were reverse transcriped to obtain cDNA which is then amplified by PCR reactions. Cell Hashtag (HTO)-containing fraction and the large cDNA library were separated by SPRI (Beckman Ampure XP beads). Purified large cDNA PCR products were used as the input for Nextera XT reactions to generate sequencing library. Purified Cell Hashtag (HTO)-containing fraction were used as input for PCR amplification using TruSeq DNA D703 primer and P5-SMARTpcr primer to generate the Cell Hashtag library. For PLZ019: mRNA molecues were captured using bead-bound oligo dT(30). Captured mRNA molecues were reverse transcriped to obtain cDNA which is then amplified by PCR reactions. The cDNA PCR reaction product was then purified with AMPure XP beads. The purified cDNA PCR product was used as the input for Nextera XT reactions to generate sequencing library. For PW073 to PW079: scRNA-seq with TotalseqA hashtag For PLZ019: scRNA-seq