GSM2586183: run1_HLA-DR+CD135+_cells; Homo sapiens; RNA-Seq
Lin(CD3/14/16/19/20/34)-CD45+CD135+HLA-DR+CD123+CD33+ single cells were sorted into each well of 384-well plates filled with 2µl lysis buffer (Triton 0.2% (Sigma) in molecular biology grade H2O (Sigma), supplemented with 0.4 U/μl protein-based RNase inhibitor (Takara)), and barcoded (400 nM (IDT)) poly-T primers. Samples were pre-incubated (3 min at 80°C) and reverse transcriptase mix (10 mM DTT (Invitrogen), 4 mM dNTPs (NEB), 2.5 U/μl Superscript III RT enzyme (Invitrogen) in 50 mM Tris-HCl (pH 8.3; Sigma), 75 mM KCl (Sigma), 3 mM MgCl2 (Sigma), ERCC RNA Spike-In mix (Life Technologies) at 1:80*107 dilution per cell) was added to each well. mRNA was reverse-transcribed to cDNA (2 min at 42°C, 50 min at 50°C, 5 min at 85°C). Excess primers were digested (ExoI (NEB); 37°C for 30 min then 10 min at 80°C) followed by a 1.2 x SPRI (Beckman Coulter) cleanup. Samples were pooled and second strands synthesized (second strand synthesis kit (NEB); 2.5 h at 16°C) followed by a 1.4 x SPRI (Beckman Coulter) cleanup. Samples were linearly amplified by T7-promoter guided in vitro transcription (T7 High Yield RNA polymerase IVT kit (NEB); 37°C for 12h). DNA templates were digested (Turbo DNase I (Ambion); 15 min at 37°C) followed by a 1.2 x SPRI (Beckman Coulter) cleanup and the RNA was fragmented (Zn2+ RNA fragmentation solution (Ambion); 1.5 min at 70°C) followed by a 2 x SPRI (Beckman Coulter) cleanup. Barcoded ssDNA adapters (IDT) were ligated to the fragmented RNAs (9.5% DMSO (Sigma), 1 mM ATP, 20% PEG8000 and 1 U/μl T4 RNA ligase I (NEB) in 50 mM Tris HCl pH7.5 (Sigma), 10 mM MgCl2 and 1mM DTT; 22°C for 2 h) and a second RT reaction (Affinity Script RT buffer, 10 mM DTT, 4 mM dNTP, 2.5 U/μl Affinity Script RT enzyme (Agilent); 2 min at 42°C, 45 min at 50°C, 5 min at 85°C) was performed followed by a 1.5 x SPRI (Beckman Coulter) cleanup Final libraries were generated by subsequent nested PCR reactions (0.5 μM of each Illumina primer (IDT; details of primers see (15)) and KAPA HiFi HotStart ready mix (Kapa Biosystems); 15 cycles according to manufacturer’s protocol) followed by a 0.7 x SPRI (Beckman Coulter) cleanup. Library quantity and quality was assessed using the Agilent 2200 tapestation system and libraries were subjected to next generation sequencing using an Illumina HiSeq1500 instrument (PE no index; read1: 61 reads (3 reads random nucleotides, 4 reads pool barcode, 53 reads sequence), read2: 13 reads (6 reads cell barcode, 6 reads unique molecular identifier))
GEO Accession
GSM2586183