GSM5856636: WT-testis longRNA rep2; Mesocricetus auratus; RNA-Seq
The single oocytes or embryos were incubated at 72°C for 3 min to release and unfold the small RNAs.For testicular RNA seq, total RNA was extracted from 21 d.p.p. golden hasmters using the RNA isolater Total RNA Extraction Reagent(VAZYME,R401-01)according to the manufacturer's protocol. For single oocyte or embryos smRNA seq, 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 Smarter-seq2 method with some modifications.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. 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.For testicular long RNA seq,DNase I was used to digest double-stranded and single-stranded DNAin total RNA, then, magnetic beads were purified to recover the reaction products, RNase H or Ribo-Zero method(Illumina, USA) was used to remove the rRNA. Purified mRNA from previous steps was fragmented into small pieces with fragment buffer at appropriate temperature. Then,first-strand cDNA was generated in First Strand Reaction System by PCR, and the second-strand cDNA was generated as well. The reaction product was purified by magnetic beads, afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to carry out end repair. The cDNA fragments with adapters were amplyfied by PCR, and the products were purified byAmpure XP Beads. Library was validating on the Agilent Technologies 2100 bioanalyzer for quality control.The double stranded PCR products above were heated denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29(Thermo Fisher Scientific, MA, USA) to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGISEQ500 platform (BGI_x0002_Shenzhen, China).For testicular smRNA seq,library was prepared with 1 μg total RNA for each sample. Total RNA was purified by electrophoretic separation on a 15% urea denaturing polyacrylamide gel electrophoresis (PAGE) gel and small RNA regions corresponding to the 18–30 nt bands in the marker lane (14-30 ssRNA Ladder Marker, TAKARA) were excised and recovered. Then the 18–30 nt small RNAs were ligated to adenylated 3' adapters annealed to unique molecular identifiers (UMI), followed by the ligation of 5'adapters. The adapter-ligated small RNAs were subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen, USA) and then several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix were performed to enrich the cDNA fragments. The PCR products were selected by agarose gel electrophoresis with target fragments 110~130 bp, and then purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The final ligation PCR products were sequenced using the BGISEQ-500 platform (BGI-Shenzhen, China).