RNA-seq testis 1ug PCR cycle 10
RNA-Seq libraries generation was similar to previously published (Zhang et al., 2012), except for using UMI-containing adaptors. Briefly, first strand cDNA was generated using ribosomal-depleted, fragmented total RNA. Resulting cDNA was incubated with a mixture of 3 sets of UMI-containing adaptors, each carrying a distinct consensus sequence as described in the results for adaptor ligation. The cDNA fragments ligated to adaptors were amplified by PCR, and the resulting libraries were sequenced using NextSeq 500 paired-end sequencing. Small-RNA-Seq generation was similar to previously described (Ghildiyal et al., 2008; Seitz et al., 2008) except for using UMI-containing adaptors. The size-selected small RNAs were first ligated to 3' adaptors containing a single consensus sequence, followed by ligation to a pool of adaptors containing one of the two distinct consensus sequences.