GSM5057609: DKO_RS_RNA-seq_Rep3; Mus musculus; RNA-Seq
Germ cells enriched preparations were prepared as follows. Decapsulated testes were treated with collagenase to digest interstitial tissue and release single tubules in suspension. The tubules were next treatment with trypsin to generate a germ cells enriched single cells suspension. The supernatants were then filtered in PVDF 100 um filter to discard tissue debris and washed with PBS. In order to obtain a better cell representation of the phenotypes observed, we included 4 different mice in each germ cells preparation. Spermatocytes and round spermatids were purified by centrifugal elutriation as described previously 3 followed by flow cytometric sorting of cells stained with Vybrant DyeCycle Green (Invitrogen, Carlsbad, CA) to obtain cell fractions with high purity. Briefly, decapsulated testes were treated with 1 mg/ml collagenase followed by treatment with 0.5 ug/ml trypsin, and the dissociated cells were used for centrifugal elutriation. Partially purified spermatocytes and the round spermatids fractions were incubated with 10 μM Vybrant DyeCycle Green for 30 min at 32°C followed by 4,6-diamidino-2-phenylindole (DAPI) staining. Cells were then sorted on a FACS-Aria (Becton Dickinson) to purify spermatocytes and round spermatids. DAPI-positive dead cells were eliminated. The purity of cells was confirmed by flow cytometry analysis of DNA content using sorted cells and by cell morphology analysis. The purification of round spermatids fraction was estimated to be approximately 93.7% ± 2.3%. The purification of spermatocytes was estimated to be approximately 81.5 ± 3.7%, based on the immunostaining with anti-SYCP3 abs. The expression of genes specific for spermatocytes and round spermatids was confirmed by RNA-seq.  For Chip-seq, germ cells enriched preparations were obtained as described before.  60 million cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by quenching with 125 mM glycine for 10 min, washed twice with 1x PBS, and resuspended in ChIP lysis buffer (150 mM NaCl, 1% Triton X‐100, 0.1% SDS, 20 mM Tris–HCl pH8.0, 2 mM EDTA). Chromatin was sheared to an average length of 200–500 bp using a Bioruptor sonication. After overnight incubation with DiaMag magnetic beads (Diagenode, Inc.) and antibodies, precipitated chromatin was then washed, decrosslinked, and digested with proteinase K.  The resulting DNA was purified using phenol/chloroform/isoamyl alcohol. DNA concentration was assessed with Quant‐it PicoGreen dsDNA kit (Invitrogen) and 5–10 ng was used to generate sequencing libraries. For CTCF Chip-seq, we used an equimolar mix of CTCF C-terminus commercial antibody were used (anti-CTCF B-5(sc-271514) and C-20 (sc-15914)) from Santa Cruz; Anti-CTCF (D3H2) from cell signaling: anti-CTCF (07-727) from Millipore: anti-CTCF (70303) from Abcam: anti-CTCF (A300-543A) from Bethyl and anti-CTCF (NB500-177 and NB500-194) from Novus. For Pol2 Chip-seq, we used the Anti-Pol2(4H8) antibodies from Abcam (ab5408). For H3K4m3 Chip-seq, we used the  Anti-H3 (tri methyl K4) antibodies from Abcam (ab8580) For RNA-seq Ion Torrent libraries,  rRNA-depleted RNA from 3 independents germ cells preparations (4 mice in each preparation) was prepared using the RiboMinus Eukaryote System v2 kit (Life Technologies) according to the manufacture recommendations. For RNA-seq Ion Torrent libraries, 500 ng of rRNA-depleted RNA was used for library preparation using the Ion Total RNA-seq v2 kit. The enriched libraries were diluted to a final concentration of 11 pM and subjected to sequencing from a single end in Ion Proton Sequencer.For RNA-seq from spermatocytes and round spermatids, total RNA was extracted from cells using Trizol (Life Technologies) according to the protocol provided by the manufacturer. The RNA quality was assessed using the Agilent Bioanalyzer 2100. The RNA sequencing library preparation and sequencing procedures were carried out according to Illumina protocols with minor modifications. Briefly, poly(A)-mRNA was purified from 5 μg of RNA with streptavidin-coated magnetic beads. After chemical fragmentation, mRNA fragments were reverse-transcribed and converted into double-stranded cDNA. Following end repair and A-tailing, paired-end adaptors were ligated to the ends of the DNA fragments. The ligated products were amplified with 18 cycles of PCR followed by purification using AMPure beads (Beckman Coulter). The enriched libraries were diluted to a final concentration of 5 nM. Each sample was subjected to 75 cycles of sequencing from a single end in Illumina Hiseq2000 Sequencer.  ChIP DNA was amplified using TruSeq ChIP Sample Preparation Kit (Illumina, Inc., USA). Briefly, the immunoprecipitated material was end-repaired, A-tailed, ligated to the sequencing adapters, amplified by 15 cycles of PCR, and size selected (200–400 bp) followed by single end sequencing on an Illumina Genome Analyzer according to the manufacturer's recommendations.