title: GSM2825939: TE_H3K27me3_rep3; Bos taurus; ChIP-Seq
library_construction_protocol: 1µl ProtG Dynabeads (Thermo Fisher Scientific) were bound with 0.25µg of corresponding antibody (H3K4me3 (07-473, Millipore), H3K27me3 (07-449, Millipore)) in 5µl of complete immunoprecipitation (IP) buffer (20mM Tris-HCl pH 7.4, 2mM EDTA, 150mM NaCl, 0.1% Triton X-100) at RT for 2 h followed by two washes using IP buffer. The density of cultured K562 or H1299 cells was determined using haemocytometer. Cells were spun down and resuspended in PBS at a density 100 or 1,000 cells per 0.5µl. Subsequently 0.5µl of lysis-restriction mix (1µl FD (FastDigest) buffer combined with 3.75µl of 2x nuclear lysis buffer (20mM TrisHCl, pH 7.4, 20mM NaCl, 6mM MgCl, 0.2% NP-40)) and 0.25µl 4x restriction enzyme mix (AluI #FD0014, SaqAI#FD2174, HinfI#FD0804, MvaI#FD0554, all FastDigest enzymes from Thermo Scientific) was added to the cells and incubated 15 min on ice and thereafter 5 min at 37¡C. Next, 1µl of 0.2% NaDOC, 0.2% TritonX-100 with protease inhibitors was added to the samples and incubated 15 min on ice after which 8µl of IP buffer (20mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM EDTA and 1%TritonX-100) and 1µl of ProtG Dynabeads (Thermo Fisher Scientific) prebound with corresponding antibody was  added to the samples. Chromatin immunoprecipitation was performed at 4¡C for four hours with end-over end mixing. After IP, beads were washed twice with 100µl of following buffers: low salt washing buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl (pH 8.1)), high salt washing buffer, IP buffer and 20mM TrisHCl, pH 7.4. Tagging was performed by resuspending the beads in 2.5µl of transposase mix (prepared by mixing 5µl of 2x buffer with 4µl mQ and 1µl Transposase) (Illumina Nextera kit) and incubation of 1 min at 37¡C. Beads were washed once with 100µl of low salt washing buffer, once with 20mM TrisHCl pH 7.4 and resuspended in 5µl of 20mM TrisHCl, pH 7.4. 16 cycles of PCR was performed using bead bound DNA as a template by mixing 5µl of beads with 2,5µl of 5µM forward and reverse primers and 10µl of 2x NEBNext enzyme. The following PCR program was used: (72C 5 min, 98C 2 min, 98C 10 sec, 63C 10 sec, 7¡C 1 min, repeat steps 3-5 15 times, hold at 4C). PCR products were purified with Agencourt RNA XP magnetic beads (Coulter Beckman), eluted in 10µl of TrisHCl pH 7.4 followed by DNA quantification using Nanodrop. The resulting library was subjected to 50 or 75bp paired end Illumina sequencing using either HiSeq2500 or NextSeq 500 platforms. RAT-ChIP-seq with bovine blastocysts was performed as described above except that after dissection the cells were collected in 3µl of embryo medium. Lysis/restriction buffer was prepared by combining 4.75µl of 10x NL (100mM TrisHCl, pH 7.4, 100mM NaCl, 30mM MgCl) buffer, 4.75µl of 10x FastDigest buffer and 0.5µl of mix of 4x restrictases. 0.75µl of lysis/restriction buffer was added to 3µl of cells and incubated 15 min on ice and 5 min at 37¡C. Next, 1µl of mix of 0.5% NaDOC, 0.5% TritonX100 with protease inhibitor cocktail was added to the samples and incubated 15 min on after which 15µl of IP buffer (20mM Tris-HCl pH 7.4, 2mM EDTA, 150mM NaCl, 0.1% Triton X-100) was added and sample was divided into 2 tubes, 10µl each for subsequent IP with Dynabeads bound with corresponding antibodies. Library was constructed using tagging resuspending the beads in 2.5µl of transposase mix (prepared by mixing 5µl of 2x buffer with 4µl mQ and 1µl Transposase) (Illumina Nextera kit) and incubation of 1 min at 37¡C. Beads were washed once with 100µl of low salt washing buffer, once with 20mM TrisHCl pH 7.4 and resuspended in 5µl of 20mM TrisHCl, pH 7.4. 16 cycles of PCR was performed using bead bound DNA as a template by mixing 5µl of beads with 2,5µl of 5µM forward and reverse primers and 10µl of 2x NEBNext enzyme. The following PCR program was used: (72C 5 min, 98C 2 min, 98C 10 sec, 63C 10 sec, 7¡C 1 min, repeat steps 3-5 15 times, hold at 4C). PCR products were purified with Agencourt RNA XP magnetic beads (Coulter Beckman), eluted in 10µl of TrisHCl pH 7.4 followed by DNA quantification using Nanodrop. Library was constructed using tagging with Tn5 Transposase (Illumina Nextera kit)
attributes.tag: GEO Accession
attributes.value: GSM2825939