Truseq cluster kit v3 cbot hs

Total RNA was isolated from each sample using the TRIzol regent (Invitrogen, Carlsbad, CA, USA). The purity, concentration, and integrity of the RNA were checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. The RNA integrity number (RIN) of all samples was >6.5, OD260/280 ≥ 1.8.
Approximately 3 µg of RNA was used per sample to construct a complementary DNA (cDNA) library, according to the following procedures: the ribosome RNA (rRNA) was removed and strand-specific RNA-Seq libraries were subsequently using rRNA-depleted RNA. After RNA fragmentation, the double-stranded cDNA was synthesized by replacing dTTPs (deoxythymidine triphosphate) with dUTPs (deoxyuridine triphosphate) in a reaction buffer used for second-strand cDNA synthesis. The resulting double-stranded cDNA was ligated to adaptors, after being end-repaired and A-tailed. Single-strand cDNA was then obtained using USER (Uracil-Specific Excision Reagent) Enzyme (NEB, Ipswich, UK). Finally, a polymerase chain reaction (PCR) was performed to enrich the cDNA libraries. Sequencing was performed on an Illumina Hiseq 2500 instrument using the TruSeq Cluster Kit v3-cBot-HS (Illumina, San Diego, CA, USA) to generate 150 bp paired-end reads.

Total RNA was isolated using Trizol reagent (Takara Inc., Dalian, China), according to the manufacturer’s instructions. The sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer’s recommendations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq Cluster Kit v3-cBot-HS (Illumia, Bologna, Italy), according to the manufacturer’s instructions. The library preparations were sequenced on an Illumina Nova platform and 150 bp paired-end reads were generated. Differential expression analysis was performed using DESeq2 R package (1.16.1). The ClusterProfiler R package was used to perform the statistical enrichment of differential expression genes in KEGG pathways and Gene Ontology (GO).

Total RNA was extracted from GC-1 cells and transfected with Prmt7 siRNA or NC siRNA using a TRIzol reagent (Invitrogen). The RNA purity was examined via NanoPhotometer^® spectrophotometer (IMPLEN, Westlake Village, CA, USA). The RNA concentration was measured with a Qubit^® RNA Assay Kit in a Qubit^® 2.0 Flurometer (Life Techonologies, Carlsbad, CA, USA). RNA integrity was tested with a RNA Nano 6000 Assay Kit of Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA).
Small RNA libraries were derived from 3 μg RNA per sample of spermatogonia for each group (Prmt7 siRNA-transfected group and NC siRNA-transfected group) in triplicate. Sequencing libraries were generated using an NEB-Next^® Multiplex Small RNA Library Prep Set for Illumina^® (NEB, Ipswich, MA, USA), following the manufacturer’s protocols. The index codes were added to attribute sequences to each sample. Then, the clustering of index-coded samples was performed on a cBot Cluster Generation System using a TruSeq Cluster Kit v3-cBot-HS (Illumina), based on the manufacturer’s instructions. Thereafter, the libraries were sequenced on an Illumina Hiseq 2500 platform. Fifty bp single-end reads were generated (Novogene, Beijing, China). The miRNA sequencing raw data were deposited in NCBI with BioProject accession number PRJNA858498.