Unambiguously mapped reads were first used to generate exon counts andthen transcript or gene counts. Feature counts were normalized using the RPM (read per million aligned reads) method, and no adjustment to gene/transcript size was made because our protocol has a limited coverage of 0.5–3 kb from the 30 end of the transcripts. An alternative analysis was used for alignments that were not aligned to their full length, where reads were aligned to a reference containing exon-exon junctions, using 42 bases on each side for junctions, allowing up to four mismatches for the full length of the read (50 bases)
The reads obtained from each cell were matched to the Mouse genome (mm 9, NCBI Build 37) and reads that aligned uniquely were used in the downstream analysis.
cDNA PCR amplification followed by SOLiD sequencing
The cells were obtained by microdissection and manual picking using glass capillaries.
Direct single cell mRNA amplification