GSM8149105: DP.mTEClo.DonorB; Homo sapiens; RNA-Seq
Tissue was minced with scissors and lymphocyte-rich supernatant was removed, then thymic fragments were digested with collagenase and dispase. EpCAM+ cells were magnetically enriched (Donors A, B, and E) or CD45+ cells were magnetically depleted (Donors C and D), and cells were stained with fluorescent antibodies. The cells were double-sorted directly into 5 μl TCL (Qiagen) buffer containing 1% 2-mercaptoenthonol (Sigma). Total RNA was purified using RNAClean XP beads (Beckman Coulter). Polyadenylated mRNA was selected using an anchored oligo(dT) primer (5′–AAGCAGTGGTATCAACGCAGAGTACT30VN-3) and then reverse transcribed to cDNA. First-strand cDNA was subjected to limited PCR amplification followed by Tn5 transposon-based fragmentation using the Nextera XT DNA Library Preparation Kit (Illumina). Libraries were constructed following the Smart-seq2 RNA-seq library preparation protocol.