alias: E-MTAB-69321544800826:Sample 11_p
title: Illumina HiSeq 2500 paired end sequencing; CUT&RUN chromatin profiling of H3K4me3, H3K27ac and H3K9me3 histone marks in spermatids and spermatocytes
design: CUT&RUN chromatin profiling of H3K4me3, H3K27ac and H3K9me3 histone marks in spermatids and spermatocytes
library_construction_protocol: Spermatogenic cell populations were isolated from adult mouse testes as described in Ernst et al. (2016). In brief, the albuginea was removed and tissue was incubated in dissociation buffer containing 25mg/ml Collagenase A, 25mg/ml Dispase II and 2.5mg/ml DNase I for 30 minutes at 37C. Enzymatic digestion was quenched with Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% Fetal calf serum (FCS, 10270106, Gibco). Cells were resuspended at a concentration of 1 million cells per ml and stained with Hoechst 33342 (H3570, ThermoFisher Scientific) at a final concentration of 5ug/ml for 45 minutes at 37C. Cells were resuspended in PBS containing 1% FCS and 2mM EDTA and propidium iodide was added to a final concentration of 1ug/ml prior to sorting. Cells were sorted on an Aria IIu cell sorter (Becton Dickinson) using a 100um nozzle. Hoechst was excited with a UV laser at 355nm and fluorescence was recorded with a 424/44 filter (Hoechst blue) and 675LP filter (Hoechst red). Primary spermatocytes (4N) and round spermatids (1N) were sorted and collected in PBS containing 1% FCS and 2mM EDTA. Cells were spun down at 600g for 3 minutes in swinging-bucket rotor and washed twice with 1.5 mL Wash buffer (20mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.5mM Spermidine and 1X cOmplete™ EDTA-free protease inhibitor cocktail (Roche)). During the cell washes, concanavalin A-coated magnetic beads (Bangs Laboratories, cat. No BP531) (10 ul per condition) were washed twice in 1.5 mL Binding Buffer (20mM HEPES-KOH (pH 7.5), 10mM KCl, 1mM CaCl, 1mM MnCl2) and resuspended in 10 ul Binding Buffer per condition. Cells were then mixed with beads and rotated for 10 minutes at room temperature (RT) and samples were split into aliquots according to number of antibodies profiled per cell type. We used 20.000-30.000 spermatocytes and 40.000-60.000 spermatids per chromatin mark. Cells were then collected on magnetic beads and resuspended in 50ul Antibody Buffer (Wash buffer with 0.05% Digitonin and 2 mM EDTA) containing one of the following antibodies in 1:100 dilution: H3K4me3 (Millipore 05-1339 CMA304, Lot2780484), H3K27ac (Abcam, ab4729, Lot GR3187598-1) and H3K9me3 (Abcam, ab8898, Lot GR306402-1). Cells were incubated with antibodies for 10 minutes at RT and then washed once with 1mL Digitonin buffer (Wash buffer with 0.05% Digitonin). For the mouse anti-H3K4me3 antibody, samples were incubated with a 1:100 dilution in Digitonin buffer of secondary rabbit anti-mouse antibody (Invitrogen, A27033, Lot RG240909) for 10 minutes at RT and then washed once with 1 mL Digitonin buffer. Samples were then incubated with 700 ng/ml Protein A-MNase fusion protein (kindly provided by Steven Henikoff) for 10 minutes at room temperature followed by two washes with 1mL Digitonin buffer.  Cells were resuspended in 100ul Digitonin buffer and cooled down to 4C before addition of CaCl2 to a final concentration of 2mM. Targeted digestion was performed for 30 minutes on ice until 100ul of 2X STOP buffer (340mM NaCl, 20mM EDTA, 4mM EGTA, 0.02% Digitonin, 250mg RNase A, 250ug Glycogen, 15pg/ml yeast spike-in DNA(kindly provided by Steven Henikoff)) were added. Cells were then incubated at 37C for 10 minutes to release cleaved chromatin fragments, spun down for 5 minutes at 16,000g at 4C and collected on magnet. Supernatant containing the cleaved chromatin fragments were then transferred and cleaned up using the Zymo Clean & Concentrator Kit.  Library preparation was performed using the ThruPLEX DNA-Seq Library Preparation Kit with a modified Library Amplification programme: Extension and cleavage for 3 minutes at 72C followed by 2 minutes at 85C, denaturation for 2 minutes at 98C followed by four cycles of 20 seconds 98C, 20 seconds 67C and 40 seconds 72C for the addition of indexes. Amplification was then performed for 12-14 cycles of 20 seconds 98C and 15 seconds 72C. Average library size was tested using an Agilent Tapestation DNA1000 High Sensitivity Screentape and quantification was performed using the KAPA Library Quantification Kit.
identifiers.namespace: EMBL-European Bioinformatics Institute
identifiers.id: E-MTAB-69321544800826:Sample 11_p