GSM2344959: Sperm-Cd-1; Mus musculus; RNA-Seq
Total RNAs were extracted from the purified sperm and testes samples using RNeasy® Plus Micro (Qiagen, Duesseldorf, Germany) according to the manufacturer’s instructions with some modifications. We added 1 ml of buffer RLT (a guanidine-thiocyanate lysis buffer supplemented with 10 mM DL-Dithiothreitol) to the sperm pellet and 10 mg testes tissues, and then homogenized the sample by passing the samples through a 20-G needle attached to a 1-ml syringe 10 to 15 times. After total RNA was bound to the membrane and other contaminants were efficiently washed away, on-column DNase digestion was performed to eliminate trace amounts DNA contamination. The concentration and integrity of the total RNA samples were evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA) and a 2100-Bioanalyzer with the RNA 6000 Nano Chip (Applied Biosystems, Carlsbad, CA, USA), respectively. RNase H protocol was performed to removal rRNA as previously described (Adiconis et al. 2013). The DNase digested RNA was purified with AMPure Beads and then fragmented to about 140–160 nt. The RNA fragments were further converted into a DNA library through end repair, adaptor ligation, reverse transcription circularization, and PCR amplification. The library was further purified with AMPure Beads and validated using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., CA, USA) and ABI StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA). The qualified library was sequenced by Illumina Illumina HiSeqTM 4000.