GSM7776975: Donor 2 iBET151 scRNA-seq HIV latency reversal; Homo sapiens; RNA-Seq
Nuclei were isolated following the 10X Genomics protocol (CG000365 • Rev C) with slight modifications. Cells were harvested, washed once with ice-cold DPBS-0.5% BSA containing 0.2 U/μl RiboLock RNase inhibitor (Thermo Fisher, cat. no. EO0382), counted and viability detected by Trypan blue (Thermo Fisher, cat. no.15250061). Dead cells were first removed using Dead Cell Removal Kit (Miltenyi Biotec, cat. No. 130-090-101). For each sample, 1×106 cells were pelleted by centrifugation at 500g, 4 °C, 5 min. The cells were resuspended in 100μl of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM sodium chloride, 3 mM magnesium chloride, 0.01% Tween-20 (Sigma, cat. no. 655205-250ML), 0.01% nonidet P40 substitute (Sigma, cat. no. I8896), 0.01% Digitonin (Promega, cat. no. G9441), 1 U/μl RiboLock RNase inhibitor, 1% BSA and 1mM DTT) and incubated on ice for 3 min. The nuclei were then pelleted and washed with 1 mL ice-cold wash buffer (10 mM Tris-HCl, pH 7.4, 10 mM sodium chloride, 3 mM magnesium chloride, 0.01% Tween-20, 1% BSA, 1mM DTT and 1 U/μl RiboLock RNase inhibitor) and re-suspended in diluted nuclei buffer (1X nuclei buffer, 1mM DTT, 1 U/μl RiboLock RNase inhibitor). Construction of single cell multiome libraries was performed using a 10X Genomics Chromium controller and a single cell multiome ATAC + Gene expression kit following manufacturers protocol. Nuclei were first isolated following the 10X Genomics protocol. Then, the intact nuclei suspensions were subjected to Tn5 transposition in bulk, followed by barcoding using GEM (Gel Beads-in-Emulsion) beads. Silane magnetic beads were used to purify the barcoded products from the post GEM-RT reaction mixture. Incubation of the GEMs produces barcoded DNA from the transposed DNA (for scATAC-seq) and barcoded, full-length cDNA from poly-adenylated mRNA (for scRNA-seq). Barcoded transposed DNA and barcoded full-length cDNA were then pre-amplified by polymerase chain reaction (PCR) to facilitate library construction. The pre-amplified product was used as input for both ATAC and gene expression (GEX) library construction. P5 and P7 indexes were added to the pre-amplified transposed DNA for ATAC-seq library. cDNA amplification, enzymatic fragmentation followed by end Repair, A-tailing, adaptor Ligation, and PCR were performed to incorporate P5, P7, i7 and i5 sample indices, and TruSeq Read 2 (read 2 primer sequence) for gene expression libraries. The libraries were quantified using an Agilent Tapestation 4200 and the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen, #Q33230). Pooled samples from ATAC and RNA libraries were sequenced using paired-end, single-index (ATAC-seq) and dual index (RNA-seq) sequencing on a NextSeq 2000 instrument (Illumina). For GEX libraries, the read format was: Read 1 - 28 cycles; Read 2 - 90 cycles; i7 - 10 cycles; i5 – 10 cycles.  For ATAC libraries the read format was: Read 1 – 50 cycles; Read 2 – 49 cycles; i7 – 8 cycles, i5 – 16 cycles. Paired end reads of pooled libraries were demultiplexed prior to downstream analysis. Paired end, dual indexing