GSM6895477: thymocytes, CD24-TCRb-, Bcl11b D/N440K, rep.1; Mus musculus; RNA-Seq
RNA was isolated using RNAClean XP beads (x2.2 vol., Beckman Coulter) directly from the lysis buffer, and was eluted into 10 uL of water containing RNase inhibitor (4u/uL RNasinPlus RNase Inhibitor, Promega). RNA-seq libraries were prepared by applying a RamDA-seq method for single-cell RNA-seq (Hayashi et al., Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs, 2018) with some modifications. The RNA was denatured by incubating at 65C for 5min, then placed on ice and treated with DNaseI (0.1U/uL DNase I, Amplification Grade, Invitrogen) in 0.25xRT Buffer (PrimeScript RT reagent Kit, Takara) at 30C for 15min. Then cDNA was synthesized in 30uL of 1xRT Buffer containing RT Enzyme Mix (1.5uL of PrimeScript RT Enzyme Mix I, Takara), oligo(dT)-RT primer (0.2uM Oligo(dT)18 Primer, Thermo Scientific), T4 gene 32 protein (0.033mg/mL, NEB) and 1st-NSR primer for mouse (3.3uM, SIGMA custom DNA oligos) by incubating 10min at 25C, 10min at 30C, 30min at 37C, 5min at 50C and 5min at 94C. Next, the second strand was synthesized by adding 5uL of 10x NEB buffer2 (NEB), 1.25uL of dNTP mix (10mM each nt, NEB), 1.5uL of Klenow Fragment (3'→5' exo-, NEB), 5uL of 2nd-NSR primer for mouse (100uM, SIGMA custom DNA oligos), and 7.25uL of DW and incubating 60min at 16C followed by 10min at 70C. The double-stranded cDNA (ds-cDNA) was purified using the AMPure XP beads (x2 vol., Beckman Coulter) and quantified using Qubit (Qubit dsDNA HS Assay Kit, Invitrogen). Then ds-cDNA less than 1ng was applied for Tn5 tagmentation in a 25uL reaction with a homemade Tn5 enzyme prepared as previously described (Picelli et al., Tn5 transposase and tagmentation procedures for massively scaled sequencing projects, 2014 and Hennig et al., Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol, 2018). DNA was purified using AMPure XP beads (x1.7 vol.) and amplified by 10~15 cycles of PCR using indexed primers and KAPA HiFi DNA Polymerase (Roche). The libraries were purified using AMPure XP beads (x1.2 vol), quantified by MultiNA (SHIMADZU) and pooled for next-generation sequencing.