HDMYZ cells were collected with Trizol reagent (Invitrogen) following manufacturer's protocol. Small RNAs of 15-40 bases were gel-purified from 10 ug total RNA The TruSeq Small RNA Sample Prep Kit (Part# RS-200-0012) is used following TruSeq Small RNA Sample Preparation Guide. The protocol takes advantage of the natural structure common to most known microRNA molecules. Most of the mature miRNA/piRNA have a 5’ phosphate and a 3’ hydroxyl group as they were created. The RNA 3’ adaptor is specifically modified to target small RNAs that have a 3’ hydroxyl group. The adapters are ligated to each end of the RNA molecule and an RT reaction is used to create single stranded cDNA. The cDNA is then PCR amplified using a common primer and a primer containing one of 48 index sequences. The introduction of the index sequence at the PCR step separates the indexes from the RNA ligation reaction. This design allows for the indexes to be read using a second read and significantly reduces bias compared to designs which include the index within the first read. After amplification the cDNA construct are further gel purified and the final library are validated by an Agilent Technologies 2100 Bioanalyzer using a DNA‐1000 chip. The multiplexed samples can be pooled in equimolar amounts. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2000 following the manufacturer's protocols. TruSeq Small RNA Library
GEO Accession
GSM1464055