Single-Cell RNA-Seq Library Preparation

Cells were sorted and collected directly into 20μL of lysis buffer containing Nuclease-free water (Ambion AM9930), 0.2% Triton and 1/20 RNAse inhibitor. We generated full-length cDNA from 3.4 μL of cell lysate according to the Smarter2 technology as described (Picelli et al., 2013 (link)). 500 pg of cDNA was tagmented with Illumina’s Nextera XT sample prep kit (Illumina Inc., U.S.A) and converted into sequencing libraries. After pooling, single-read 43bp sequencing was performed on Illumina HiSeq2000 using the Truseq v3 sequencing chemistry (Illumina Inc., U.S.A). TopHat2 version 2.0.10 (Kim et al., 2013 (link)) was used to align the reads to the mouse reference genome (mm10). Gene expression values for RefSeq transcripts were calculated as TPMs using Cufflinks (Trapnell et al., 2012 (link)) (2.1.1). Raw reads were counted with the summarizeOverlaps function with the GENCODE M19 gene annotation using the union mode from the Bioconductor Genomic Alignments package (Lawrence et al., 2013 (link)) (v1.18.1).