PNLDC1 KO Round spermatids total RNAs rep1
Total RNA from sorted germ cells was extracted using mirVana miRNA isolation kit (Thermo Fisher, AM1560) and used for library preparation as described previously (Zhang et al., 2012) with several modifications including addition of the ERCC spike-in mix to enable absolute quantification of RNAs and the use of unique molecular identifiers to eliminate PCR duplicates (Fu et al., 2018). Briefly, before library preparation, 1 l of 1:100 dilution of ERCC spike-in mix 1 (Thermo Fisher, 4456740, LOT00418382) was added to 0.51 g total RNA to enable absolute quantification of mRNA. For ribosomal RNA depletion, RNA was hybridized in 10 l to a pool of 186 rRNA antisense oligos (0.05 M each) in 10 mM Tris-Cl (pH 7.4), 20 mM NaCl, heating the mixture to 95C, cooling it at ?0.1C/sec to 22C, and incubating at 22C for 5 min. RNase H (10U; Lucigen, H39500) was added and the mixture incubated at 45C for 30 min in 20 l containing 50 mM Tris-Cl (pH 7.4), 100 mM NaCl, and 20 mM MgCl2. RNA was further treated with 4U DNase (Thermo Fisher, AM2238) in 50 l at 37C for 20 min. After DNase treatment, RNA was purified using RNA Clean & Concentrator-5 (Zymo Research, R1016). RNA-seq libraries for three independent biological replicates were sequenced using a NextSeq 500 (Illumina) to obtain 75 + 75 nt, paired-end reads.
SUB4468410
Zamore74