GSM5208889: KO-secondary longRNA rep1; Mesocricetus auratus; ncRNA-Seq
Ten MII oocytes of WT and PIWIL1 mutant golden hamster were lysed with lysis buffer composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 0.1 U/μl RNase Inhibitor (Thermo Fisher Scientific, Massachusetts, USA) and 0.4 U/μl Proteinase inhibitor cocktail (Sigma, USA) on ice for 10 min. Dynabeads Protein G (Thermo Fisher Scientific, Massachusetts, USA) were washed once with lysis buffer and coupled with 1.5 μg of homemade anti-PIWIL1 or rabbit IgG (Millipore) at RT for 40 min. Oocyte lysates were mixed with antibody-coupled beads and rotated gently at 4°C for 5 h. The beads were then washed three times with lysis buffer and once with PBS. Total RNA from the beads was eluted by adding 4 μl of water and incubating at 72°C for 5 min. The supernatant containing RNA was recovered on a magnetic holder and then used for cDNA library construction. For testis, fresh tissues were homogenized in lysis buffer using Precellys 24 tissue homogenizer (Bertin, USA) and immunoprecipitation was performed as described above. Small RNA libraries were constructed using the single-cell small RNA library construction method as described above. The single oocytes or embryos were incubated at 72°C for 3 min to release and unfold the small RNAs. After 3' adapter ligation, 5U of lambda exonuclease and 25U of 5' deadenylases, which performed better than the combination of RecJf and 5' deadenylases, were used to remove the excess 3' adaptor. Then, a 5' adapter was ligated and small RNAs were reverse transcribed. Pre-amplification was performed and then 1 μl of amplified product was used as a template for final amplification. The amplified libraries were separated on a 6% polyacrylamide gel and 130-160 bp DNA fragments were sliced to recover the library of small RNAs. Long RNA transcriptome libraries was performed following the previously described Smarter-seq2 method with some modifications. In brief, single oocytes or embryos were incubated at 72°C for 3 min to release the RNAs. For every single oocyte or embryo, 0.125 μL 1/100,000 dilution of the ERCC RNA Spike-In Mix (Invitrogen, Cat# 4456740) was used. Following reverse transcription and PCR pre-amplification, cDNA libraries were purified by Agencourt Ampure XP beads (0.8:1 ratio) (Beckman Coulter, Cat#A63881) and 2 ng of purified cDNA was used for a tagmentation reaction with Tn5 transposase as described previously (53). Amplified libraries were purified by Agencourt Ampure XP beads (1:1 ratio). The small and long RNA libraries were sequenced on a HiSeq X Ten instrument (Illumina, USA) with 2 × 150 run cycles.