GSM6889986: stage III, Rep#2, Exp #2, scRNAseq; Homo sapiens; RNA-Seq
For scRNA-seq, the neuro-spheres were digested by TrypLE Enzyme at 37℃ for 10 minutes and dissociated into single cells. After 35-µm cell sieve filtering, the digested cells were sorted by FACS to collected EGFP-positive cells, which were then concentrated by centrifuge at 400 g for 5 minutes. All the sorted EGFP-positive cells were used to construct single cell library using Chromium Single Cell 3' Reagent Kits (v2 and v3) following the manufacturer's guidelines (10X Genomics). scRNA-seq library was performed according to the manufacter's instructions (single cell 3' v2 and v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. For barcoded viral library, we designed primer pairs with illumina adaptors to amplify the random barcode sequences from the retroviral barcode library plasmid pool (Forward, AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTATTCGGAGGACGACCCTATTTG; Reverse, CAAGCAGAAGACGGCATACGAGATAGGATTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGATCACTCTCGGCATGGACG) with the following PCR program: 1, 98 °C, 3 min; 2, 98 °C, 10 s; 3, 60 °C, 10 s; 4, 72 °C, 25 s; 5, repeat steps 2–4 15 times; 6, 72 °C, 3 min; 7, 4 °C, hold. The PCR products were purified by 0.85× left-sided size selection using Ampure XP bead (Beckman Coulter, catalogue number A63880) and then sequenced on Illumina HiSeq PE150. For cell lineage barcode recovered from single cell transcriptome, following the reverse transcription of mRNA and first strand cDNA amplification, 50 ng of the unfragmented cDNA libraries were used as templates to specifically amplify cell barcodes and virus barcodes with primers pairs: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC and CAAGCAGAAGACGGCATACGAGATAGGATTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCATGGACGAGCTGTACAAATAA, using the following program: 1, 98 °C, 3 min; 2, 98 °C, 10 s; 3, 60 °C, 15 s; 4, 72 °C, 1 min; 5, repeat steps 2–4 20 times; 6, 72 °C, 3 min; 7, 4 °C, hold. The PCR products were then purified by 0.8-0.6× dual-sided size selection using Ampure XP bead and sequenced using Illumina HiSeq PE150.