PNLDC1 KO Primary Spermatocytes MIWI IP small RNAs rep2 oxidized
Small RNA libraries were constructed as described (Han et al., 2015a) with several modifications. Briefly, before library preparation, a set of 18 synthetic RNA oligonucleotides was added to each RNA sample to enable absolute quantification of small RNAs (Table S2A). To reduce ligation bias, a 3? adaptor with three random nucleotides at its 5? end (5?-rApp NNN TGG AAT TCT CGG GTG CCA AGG /ddC/-3?) and ligation buffer containing 20% PEG-8000 (f.c.) were used. After 3? adaptor ligation, RNA was purified by 15% urea polyacrylamide gel electrophoresis (PAGE), selecting for 1555 nt small RNAs (i.e., 4080 nt with 3? adaptor). Small RNA-seq libraries for two independent biological replicates were sequenced using a NextSeq 500 (Illumina) to obtain 75 nt, single-end reads.
SUB4468410
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