GSM7110046: TUH93 CTL; Homo sapiens; RNA-Seq
Purification of hCD45+CD33+ from PDX : After red blood cells lysis with EL buffer (Qiagen, 79217), 10x106 cells from mice bone marrow of PDXs treated with vehicle or GILT were stained with Fixable Viability Stain 510 (BD Biosciences Cat# 564406, RRID:AB_2869572) and the fluorescent conjugated antibodies hCD45-APC (BD Biosciences Cat# 555485, RRID:AB_398600) and hCD33-PE (BD Biosciences Cat# 555450, RRID:AB_395843) in cell staining buffer (Biolegend, BLE420201) following to the manufacturer's instructions. Viable hCD45+CD33+ were sorted using BD FACSAria Fusion flow cytometer (Becton Dickinson) and gently re-suspended at 1500cells/µL in PBS1X supplemented with 0.04% Ultrapure BSA (Invitrogen, Cat# AM2616). Up to 10,000 cells per sample were encapsulated using the 10X Chromium controller (10X Genomics) following the Chromium Next GEM Single Cell 3ʹ User Guide v3.1 (10X Genomics). Briefly, cells were encapsulated into oil droplets with barcoded Gel Beads and reagents to convert mRNA into complementary DNA (cDNA). cDNAs were then amplified, fragmented and Illumina adapters were added during ligation. After performing a last index PCR, libraries quality was assessed using Tapestation 2200 (Agilent) and frozen before quantitation and sequencing. Barcoding was done on three levels: cell barcodes allow attribution of each sequence read to its cell of origin; unique molecular identifiers (UMIs) upstream to poly(d)T primers allow tagging of each original molecule to avoid amplification bias; and index allows pooling of different samples. scRNA-seq libraries were quantified with HSD1000 reagents on TapeStation 2200 system (Agilent) and pooled at equimolar concentrations within 200 to 700bp. Pool libraries were sequenced at 1.8pM 1% phiX using a NextSeq 500 sequencer (Illumina), at a sequencing depth of 40,000-50,000 reads per cell. Sequencing were configured Paired-End at 28 cycles for the first read, 91 cycles for the second one and 8 cycles for a single index.