sRNA-seq

Total RNA was extracted by following the standard protocol of TRIzol method. 10 µg total RNA was run on a 15% PAGE/7 M Urea gel (Bio-Rad) and the sRNA fraction (18–25 nt) was excised and eluted from the gel, which was subsequently cloned using the NEBNext multiplex small RNA library prep kit (NEB). Libraries were indexed during the PCR step with 12 cycles and gel size-selected and purified. Four biological replicates of libraries for each genotype were constructed. Pooled libraries were sequenced on a NextSeq 500 (Illumina). Sequencing reads were trimmed using Trim Galore! with default parameters and then mapped to the Heinz genome SL3.0 using Bowtie 1.1.1⁴⁰ with specified parameters of -m 1 and -v 0 for unique mapping and no mismatch allowed respectively. The output sam files were converted to bam files by Samtools⁴¹. The uniquely mapped bam files were used for RPKM analysis of sRNA.