cDNA library was generated using Illumina TruSeq small RNA kit (Cat#RS-200-0012) and only 3′-adapter (RA3) need to be ligated enzymatically using truncated T4 RNA ligase (NEB M0242) on to the fragmented aRNA and the 5′ ligation step for RA5-adapter was skipped (Hashimshony et. al. 2012). Adapter ligated fragmented aRNA was reverse transcribed using SuperScriptIII reverse transcriptase (Invitrogen, USA) and PCR enriched using TruSeq indices (for multiplexing) for no more than 7–11 cycles. The resulting library was size-selected using SPRISelect magnetic beads (Agencourt) to select 350–450bp fragments and paired-end sequenced for 101bp in Illumina HiSeq. No more than 32 single cells were run in one lane of HiSeq2000 generating on average ~180–200 million reads per lane.
