GSM6416224: mTEClo, neonate, WT, cell58; Mus musculus; RNA-Seq
Mouse thymus was enzymatically digested and CD45+ cells were depleted using mouse CD45 MicroBeads and a LS Column (Miltenyi Biotec). After staining with an antibody cocktail, mTEChi (DAPI-CD45-EpCAM+Ly51-MHCII-high), mTEClo (DAPI-CD45-EpCAM+Ly51-MHCII-low), and cTEC (DAPI-CD45-EpCAM+Ly51+) were sorted by FACSAria into a 96well plate containing 1uL of lysis buffer (0.3% NP40; Thermo Scientific, 1U/uL RNasinPlus; Promega in 1x Lysis Buffer, Takara) scRNA-seq libraries were prepared by a RamDA-seq method (Hayashi et al., Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs, 2018). Specifically, RNA was denatured by incubating at 70C for 90sec, 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 5min. Then cDNA was synthesized in 3uL of 1xRT Buffer containing RT Enzyme Mix (0.15uL 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 (2.7uM, 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 0.5uL of 10x NEB buffer2 (NEB), 0.125uL of dNTP mix (10mM each nt, NEB), 0.15uL of Klenow Fragment (3'→5' exo-, NEB), 0.4uL of 2nd-NSR primer for mouse (100uM, SIGMA custom DNA oligos), and 0.825uL 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 (Beckman Coulter) and applied for Tn5 tagmentation in a 5uL 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). After dissociating Tn5 protein by adding SDS (0.04% in final), tagmented DNA was amplified by 17 cycles of PCR using indexed primers and KAPA HiFi DNA Polymerase (Roche). The libraries were purified using AMPure XP beads (x1.0 vol), quantified by MultiNA (SHIMADZU) and pooled for next-generation sequencing.