GSM8322243: DC1-P4; Mus musculus; RNA-Seq
Single-cell transcriptome amplification followed the SMART-SEQ2 protocol with minor modifications. Briefly, single cells were sorted into 2.55 ul cell lysis buffer containing RNase inhibitor (40 U/ul), Triton X-100 (10%), barcode primer (5 uM), and dNTP mix (10 mM). Reverse transcription was performed using a 25-nt oligo(dT) primer anchored with an 8-nt cell-specific barcode and 8-nt unique molecular identifiers (UMIs). After first-strand synthesis, second-strand cDNA was synthesized, followed by amplification through PCR. The amplified cDNA was pooled and further amplified using biotinylated pre-indexted primers. Approximately 300 ng cDNA was sheared to ~300 bp using a Covaris S2, and the 3' end was captured using Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher). Libraries were constructed from the enriched cDNA fragments attached to the C1 beads using KAPA Hyper Prep Kits (KK8505) and NEB U-shape adaptor for ligation.