GSM8830706: scRNA-seq_ATRXKO_ery; Homo sapiens; RNA-Seq
Single live nucleated cells were sorted into each well of a cold 384-well PCR plate (FrameStar 4titude, Cat No 4ti-0384/C) containing lysis buffer with barcoded Oligo(dT)-tailed oligonucleotides as described in Rodriguez-Meira et al., 2019. The sorted plates can be stored at -80°C for up to 3 month before library processing. Retrotranscription and PCR steps were performed on the sorted plates. Briefly, proteinase in the lysis buffer was heat-inactivated at 72°C for 15 min, during which retrotranscription mix was prepared by mixing SMARTScribe retrotranscriptase (Clontech, Cat No 639537), RNase inhibitor (TaKara, Cat No 2313A), and template-switching oligo TSO-LNA (sequence: AAGCAGTGGTATCAACGCAGAGTACATrGrG+G, RNase-free HPLC purified). The RT mix was dispensed into each well after heat inactivation and first-strand cDNA was synthesised. Afterwards, the cDNA was amplified by SeqAMP (Clontech, Cat No 638509) using 21 PCR cycles. Amplified products from each plate were pooled, bead-purified, tagmented, and indexed using Tn5 enzyme (Illumina Nextera XT Kit, Cat No FC-131-1096) to construct libraries. The libraries were purified with AMPure beads, assessed on TapeStation System (Agilent), quantified using the NEBNext Library Quant Kit (NEB, Cat No E7630L), and sequenced on a NextSeq platform with 75 bp single-end reads using the NextSeq 500/550 High-output 75 Cycle Kit (Illumina, Cat No 20024906).