title: GSM2690949: d5 NGO input BS_1; Mus musculus; Bisulfite-Seq
library_construction_protocol: ChIP-bisulfite-seq was done on MNase-digested 10% input samples from the ChIP protocol, detailed in the corresponding ChIP-seq samples.  Following DNA purification from chromatin using spri purification with Sera-Mag carboxylate-modified Magnetic SpeedBeads, samples were bisulfite converted using the 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 Dynabeads M-280 Streptavidin beads. Samples are then subjected to second strand synthesis similar to the first strand synthesis, followed by 16-20 PCR amplification cycles using Phusion High-Fidelity DNA polymerase. Cells for ChIP-seq were flash frozen in nuclear lysis buffer, cells for PBAT were flash frozen in PBS The ChIP-seq libraries for all samples were prepared using an ultra-low input native ChIP protocol, as previously described 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 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.  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 from immunoprecipitated chromatin or input was purified using spri purification using Sera-Mag carboxylate-modified Magnetic SpeedBeads at a 1.8:1 ratio.  Library preparation was completed using the Diagenode MicroPlex Library Preparation kit v2 using Sanger 8-base indices for multiplexing, as per the manufacturers recommendations. For single oocyte RNA profiling, four GV oocytes from Mll2 KO and WT 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 (New England Biolabs). 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 Dynabeads M-280 Streptavidin beads. Samples are then subjected to second strand synthesis similar to the first strand synthesis, followed by 10 PCR amplification cycles using Phusion High-Fidelity DNA polymerase. ChIP-bisulfite-seq was done on MNase-digested 10% input samples from the ChIP protocol, detailed in the corresponding ChIP-seq samples.  Following DNA purification from chromatin using spri purification with Sera-Mag carboxylate-modified Magnetic SpeedBeads, samples were bisulfite converted using the 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 Dynabeads M-280 Streptavidin beads. Samples are then subjected to second strand synthesis similar to the first strand synthesis, followed by 16-20 PCR amplification cycles using Phusion High-Fidelity DNA polymerase.
attributes.tag: GEO Accession
attributes.value: GSM2690949