GSM7968797: Intraocular RB 1, scRNAseq; Homo sapiens; RNA-Seq
Fresh human RB tissue samples were immediately washed three times in ice-cold PBS after collection. Dissociation into single cells was achieved by incubation in 0.25% trypsin-0.01% EDTA (Invitrogen) at 37 °C for 5 min, gentle pipetting and flicking, and trypsin was then blocked with addition of FBS (Gibco). Dissociated cells were filtered with 70 μm cell strainer (Corning) and centrifuged at 1000 rpm for 5 min, and followed by resuspension in 200 to 500 ul ice-cold PBS. Cell counting and viability rate were determined with Countess II automated cell counter (Invitrogen) after 0.4% trypan blue staining, and the sample quality was only considered ideal for further single-cell assessment when the cell viability was higher than 90% and clustering rate was less than 1%. Samples were processed within 3 h from surgical removal to loading on the Chromium (10x Genomics) instrument. GEMs (Gel Bead-In-EMulsions) are formed by combining the prepared single-cell suspension with gel beads containing barcode information and a mixture of enzymes, which are then encapsulated by oil droplets located in microfluidic "single-cross" junctions. Valid GEMs include beads (pre-made 10x primers in beads), single cells, and Master Mix. Then, cell lysis and reverse transcription reactions were performed within the GEMs. In valid GEMs, 10x Barcode would be ligated with the cDNA product. Next, GEMs and oil droplets were broken. PCR amplification was performed using the cDNA as a template. After the amplification was completed, the quality inspection of the amplified products (the size of the amplified fragments and the yield of the amplified products) was performed. After the amplification products were qualified, the sequencing library was constructed. First, the cDNA was chemically broken into fragments of about 200~300bp; cDNA was fragmented, end-repaired and A-added; cDNA fragments were screened; the P7 adapter was connected and the sample index was introduced by PCR amplification; and finally fragment screening is performed to obtain cDNA library.