GSM2701333: Lgr6_Mouse1_SC_S21; Mus musculus; RNA-Seq
Agencourt RNA-Clean strepdavadin beads were used to precipitate nucleic acids, which were cleaned by washing with 70% ethanol.  RNA extraction step is done with Agilent Bravo Automated Liquid Handlin Platform. We used a modified version of the SmartSeq2(SS2) protocol (Picelli et al., 2013), as previously described (Shekhar et al., 2016).  Library construction protocol: Reverse transcription of polyadenylated transcripts was done by using an oligo-dT primer. A reverse transcriptase was derived from the Moloney murine leukemia virus (MMLVRT) followed by a template switching reaction that relies on the terminal-transferase activity of the MMLVRT in the presence of a template switch oligonucleotide primer (TSO). The double-stranded RT-product was PCR amplified using Kapa Ready Mix to yield the whole transcriptome amplification (WTA) product. The WTA product was cleaned up with AMPure SPRI beads and 80% ethanol, and QCed with BioAnalyzer and qubit.  Each single-cell library was individually barcoded by PCR with index primers. The barcoded single cells were pooled and sequenced on an Illumina NextSeq sequencer.