title: GSM4222507: pachytene_spermatocytes1 (PS1), RNA-seq; Sus scrofa; RNA-Seq
library_construction_protocol: Fractions with high purity of spermatogonia, pachytene spermatocytes and round spermatids were resuspended with Trizol (Invitrogen, USA) and total RNA was extracted for further analysis. RNA seq: The rRNAs were removed from total RNA by using NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instruction. NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts,USA) was used to construct RNA libraries. Library quality were confirmed by BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an Illumina Hiseq 4000 with 150 bp paired end reads. MeRIP-seq: In brief, total RNA was extracted by using TRIzol Reagent (Invitrogen, USA). Denaturing agarose electrophoresis was used for confirming RNA integrity. Then, mRNA was isolated from total RNA by using Seq-Star T M poly(A) mRNA Isolation Kit (Arraystar, MD, USA). The m6A RNA immunoprecipitation was conducted by using GenSeq™ m6A RNA IP Kit (GenSeq Inc., China) according to the manufacturer's instructions. Libraries were constructed from the samples with and without m6A immunoprecipitation by using NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., MA, USA). Library was evaluated by the BioAnalyzer 2100 system (Agilent Technologies Inc., CA, USA) and sequenced on an Illumina Hiseq 4000 with 150 bp paired-end reads.  After quality controlled by Q30, 3' adaptor and low quality reads were removed by cutadapt software (v1.9.3). Clean reads of all libraries were aligned to the reference genome (SusScr3) by Hisat2 software (v2.0.4). Methylated sites were identified by MACS software and further used for differentially methylation analysis. Gene ontology analysis were performed by the differentially methylated protein coding genes.
attributes.tag: GEO Accession
attributes.value: GSM4222507