GSM9111422: adult mouse testis RNA-Seq; Mus musculus; RNA-Seq
For RNA-isolation, a testis piece, freshly isolated or stored at -80°C was homogenized using a tissueLyser (Qiagen cat. 85220) in a 2 ml Eppendorf tube, 1 min, frequency 30.0. Total RNA prepared with Trizol (Invitrogen, Thermo Scientific, see below) was DNAse digested and further purified using the RNeasy Micro kit (Qiagen). Any residual genomic DNA was digested on column according to manufacturer's instructions, with the addition of an extra 1µl of RNase-free DNase I (Roche). The RNA was eluted using RNase-free water, quantified using the Qubit RNA HS Assay and the integrity was verified using Bioanalyzer RNA Pico chips (Agilent).  For ChIP-Seq, we isolated single cells from mouse testes. Crosslinking of approximately 1x10^6 cells was performed in PBS. Cells were washed twice with cold PBS containing 0.05% Triton X-100, pelleted, snap frozen and stored at -80°C until sonication. Cells were processed using the iDeal ChIP-Seq kit (Diagenode) according to manufacturer's instructions. Sonification was performed on a Bioruptor Pico (Diagenode) using 3 runs of 10 cycles (30 s on, 30 s off) in a 4°C water bath. Sheared chromatin was purified and the size distribution was verified using a DNA HS Bioanalyzer chip (Agilent). Approximately 200,000 cells were used for ChIP with the anti-H3K27ac (ab4729, Abcam) antibody. Approximately 150-200 ng of total RNA was used for the generation of strand-specific RNA-seq libraries using the ScriptSeq v2 (Epicentre) low input libarary preparation kit according to manufacturer's instructions. The RNA-seq libraries were quantified using the Qubit high sensitivity DNA assay (Life Technologies) and the size distribution was verified using the DNA HS Bioanalyzer chips (Agilent). Libraries were paired-end sequenced on a HiSeq 2000 (Illumina) with 2x50 bp read length.  Approximately 1-5 ng of ChIP DNA was used to generate libraries using the TrueSeq ChIP-Seq kit (Ilumina) with minor modifications. After adapter ligation, a 0.95x volume of AMPure XP beads (Beckman Coulter) was used for a single round of purification. After the addition of 1 µl primer mix (25 mM each, Primer 1: 5'-AATGATACGGCGACCACCGAG-3'; Primer2: 5'- CAAGCAGAAGACGGCATACGAG-3') and 15 µl 2x Kapa HiFi HotStart Ready Mix (Kapa Biosystems), amplification was performed for 45 s at 98°C, 5 cycles of [15 seconds at 98°C, 30 s at 63°C and 30 s at 72°C] and a final 1 min incubation at 72°C. The PCR products were purified using a 0.95x volume of AMPure XP beads. The libraries were amplified for further 13 cycles, purified using a 0.95x volume of AMPure XP beads, quantified using the Qubit DNA HS assay and the size was validated using DNA HS Bioanalyzer chips (Agilent). Libraries were sequenced on a NextSeq500 (Illumina) with 1x75 bp read length.