Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described (Brind’Amour et al. 2015) with the several modifications. Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as per recommendations in the Brind’Amour protocol. Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with EDTA. Chromatin samples were precleared with Protein A/G beads rotating at 4°C for 2 hours and antibodies were bound to beads rotating at 4°C for 3 hours. For each immunoprecipitation of ~500 cells, 250ng of anti-H3K4me3 (Diagenode ab4729), 125ng of anti-H3K27me3 (Millipore 07-449), or 125ng of anti-H3K27ac (Abcam ab4729) antibodies were used. Chromatin was added to the antibody-bound beads and rotated overnight at 4°C. This was proceeded by two low-salt washes, one high salt wash, and the DNA was then eluted from the beads at 65°C for 1.5 hours. DNA was purified using spri purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. Library preparation was completed using the MicroPlex Library Preparation kit v2 (Diagenode) using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations. Multiplexed libraries were sequenced using50bp single-end mode on the HiSeq 2500 sequencing technology (Illumina), as per the manufacturer’s recommendations. Cells for ChIP-seq were flash frozen in nuclear lysis buffer The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described (Brind’Amour et al. 2015) with the several modifications. Samples were thawed on ice and permeabilised with 0.1% Triton-X/0.1% deoxycholate in PBS on ice. MNase digestion was completed using 200U of micrococcal nuclease (New England Biolabs) in prepared digestion buffer, as per recommendations in the Brind’Amour protocol. Samples were digested at 21°C for 7.5 minutes and the reaction was stopped with EDTA. Chromatin samples were precleared with Protein A/G beads rotating at 4°C for 2 hours and antibodies were bound to beads rotating at 4°C for 3 hours. For each immunoprecipitation of ~500 cells, 250ng of anti-H3K4me3 (Diagenode ab4729), 125ng of anti-H3K27me3 (Millipore 07-449), or 125ng of anti-H3K27ac (Abcam ab4729) antibodies were used. Chromatin was added to the antibody-bound beads and rotated overnight at 4°C. This was proceeded by two low-salt washes, one high salt wash, and the DNA was then eluted from the beads at 65°C for 1.5 hours. DNA was purified using spri purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. Library preparation was completed using the MicroPlex Library Preparation kit v2 (Diagenode) using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations. Multiplexed libraries were sequenced using50bp single-end mode on the HiSeq 2500 sequencing technology (Illumina), as per the manufacturer’s recommendations. For single oocyte RNA profiling four GV oocytes from Mll2Gdf9cKO and control mice were collected for each genotype. Cells were lysed, RNA reverse transcribed and amplified according to the protocol of SMARTer Ultra Low RNA Kit for Illumina Sequencing (version 1, Clontech). Subsequently, samples were subjected to NEBNext Ultra DNA library preparation for Illumina using indexed adaptors (NEB). Resulting libraries were pooled in equimolar quantities for 75-bp single-read sequencing on Illumina HiSeq 2000, resulting in about 15-18 million reads per sample. Post-bisulfite adaptor tagging (PBAT) was used to generate whole-genome bisulfite sequencing libraries using 100 collected oocytes. Briefly, cells are lysed with 0.5% SDS in EB buffer and bisulfite treated with one-step modification procedure using the Sigma Imprint DNA Modification kit. The resulting DNA are purified using columns from Zymo EZ DNA Methylation Direct kit. First strand synthesis is then performed using Klenow Exo- with customized, streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs of random sequences (9N). This is followed by exonuclease I treatment and binding to biotin-containing beads. Samples are then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles.