title: GSM2356623: SMC1B-LAP ChIP-Seq (Input); Mus musculus; ChIP-Seq
library_construction_protocol: Single-cell suspensions of testes were obtained by removal of the tunica albuginea and mashing of the tissue followed by filtration through a 40 µm nylon mesh into PBS to remove cell clumps. For formaldehyde fixation, cells were resuspended at a density of 5 million/ml in DMEM+GlutaMAX containing 10% fetal calf serum (FCS) after additional centrifugation. Formaldehyde was added to a final concentration of 1% (w/v) and cells were crosslinked for 10 min at room temperature under slight agitation. Cells were quenched by addition of glycine to a final concentration of 125 mM for 5 min at room temperature. Crosslinked and non-crosslinked cells were centrifuged and resuspended in FACS buffer (HBSS supplemented with 20 mM HEPES (pH 7.2), 1.2 mM MgSO4, 1.3 mM CaCl2, 6.6 mM sodium pyruvate, 0.05% lactate, glutamine, and 1% fetal calf serum). Cell sorting of GFP-positive cells was performed on an ARIA II (BD Biosciences). Cells were collected into PBS and reanalyzed, counted in a Neubauer chamber, pooled after centrifugation and frozen at -80°C or directly undertaken subsequent ChIP experiments. FlowJo (Tree Star) and FACSDiva softwares were used for cell sorting and data analysis. As a first step of library preparation, purified DNA molecules of ChIP and input samples were end-repaired with the NEBnext End Repair Module (New England Biolabs) and purified using Agencourt AMPure XP Beads (Beckman Coulter). Blunt-ended fragments were then supplied with a 3’-dA overhang using the NEB- next dA-Tailing Module (New England Biolabs) and purified (see above). Subsequently, adapters (Adaptor-Oligo 1: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’, Adaptor-Oligo 2: 5’-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’) were ligated for large-scale PCR amplification using the 1x NEBnext Quick Ligation Buffer (New England Biolabs using 10x excess of DNA Adaptors, 5 μl Quick T4 DNA Ligase (New England Biolabs) at 50 μl total volume). Size selection of fragments of an adequate size range was performed after separation by agarose gel electrophoresis. After purification, large-scale amplification of library constructs was performed using the PCR Enrich Adaptor Ligated cDNA Library module (New England Biolabs) with subsequent purification. For quality control and size selection, fragments were run on a 2% agarose E-Gel (Invitrogen) and quantified using the Qubit dsDNA HS Assay Kit (Invitrogen).