alias: GSM2735962
title: GSM2735962: MCF7_emptyVector_doxInduced_rep1_WGBS; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Whole Genome Bisulfite Sequencing by MethylC-seq libraries were constructed as follows. Genomic DNA was extracted from cells using the Qiagen Blood and Tissue Kit according to the manufacturer's instructions. 1 ug of genomic DNA was spiked with 0.5% (w/w) of unmethylated lambda phage DNA for the calculations of the non-conversion rate and sheared with a Covaris S2 sonicator to an average length of 200 bp. The sheared DNA was end-repaired, A-tailed and ligated to methylated Illumina TruSeq adapters and subjected to 4 cycles of PCR amplification using KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems). TAB-seq libraries were constructed as follows. Genomic DNA was isolated as described for MethylC-seq. TAB-seq libraries were generated using the 5hmC TAB-seq kit (WiseGene) according to the manufacturer’s instructions. 5-hydroxymethylated pUC19 DNA (WiseGene) was used to estimate the protection of 5hmC by β-glucosyltransferase and unmethylated lambda phage DNA was used to estimate the bisulfite conversion rate. RNA-seq libraries were constructed as follows. RNA was extracted from cells using the Qiagen RNeasy kit. 330 ng of total RNA was used to generate libraries using the Illumina TruSeq Stranded mRNA kit according to the manufacturer's instruction, except one-third of all reaction volumes were used and the final amplification used 10 cycles of PCR. ChIP-seq libraries were constructed as follows. cells were harvested at 72 h by crosslinking in a final concentration of 1% formaldehyde. Crosslinking was stopped after 5 min by adding glycine to a final concentration of 125 mM. Crosslinked cells were washed in cold phosphate buffered saline, lysed using 1 ml low-salt IP buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%) containing protease inhibitors) and aliquoted at 1 × 10∧7 cells/ml. Cells are then sonicated to a fragment size range of 500–800 bp. Samples were then diluted in 1 ml low-salt buffer and incubated with 3 μl of anti-HA antibody (Covance, Princeton, NJ). Three-hundred microliter Sepharose A beads (GE Healthcare Life Science) were used for pull-down. ChIP-bisulfite sequening libraries were constructed as follows. Two 15 cm plates of cells were grown and doxycycline induced for three days. Cells were washed two times with 10 ml of PBS and crosslinked for 5 minutes in 50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 1mM EDTA, 1% formaldehyde. The crosslinking reaction was quenched by the addition of glycine to a final concentration of 125 mM and washed two times with phosphate buffered saline (PBS). All subsequent solutions were supplemented with a protease inhibitor cocktail from Sigma (Cat. # P8340). Cells were scraped off the plates with a rubber policeman in 10 ml of PBS and spun at 3,000 rpm for 5 minutes in a swinging bucket rotor. The cell pellets were resuspended in 10 ml of 50 mM HEPES-KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, incubated on ice for 10 minutes and centrifuged at 3,000 rpm for 10 minutes in a swinging bucket rotor. Cell pellets were washed two times by adding gently adding 10 mM Tris-HCl, pH 8.1, 200 mM NaCl, 1 mM EDTA to the cell pellets trying not to disturb the pellets and centrifuged at 3,000 rpm for 5 min. Finally the cell pellets were resuspended in 0.1% SDS, 1 mM EDTA and transferred to a Covaris TC12x12 tube. The chromatin was sheared with a Covaris S2 sonicator with the following settings: Time 12 min, duty cycle 5%, intensity 4, cycles per burst 200, temperature 4˚C, power mode frequency sweeping. Triton X-100 and NaCl were added to a final concentration of 1% and 150 mM respectively. The sheared chromatin was centrifuged at maximum speed in a microfuge for 15 minutes at 4˚C and the supernatant was transferred to a new tube. 2 ul of anti-H3K4me3 (Diagenode, Cat. # C15410003) or 4 ul of anti-phospho-Ser5 RNA polymerase antibody (Active Motif, Cat. # 39233) was added and incubated overnight at 4˚C. 30 ul of Protein G Dynabeads (Life Technologies) was added and incubated on a tube rotator for 90 minutes at 4˚C. The beads were washed two times with 20 mM HEPES-KOH, pH 7.9, 0.1% SDS, 150 mM NaCl, 1% Triton X-100 2 mM EDTA, two times with 20 mM HEPES-KOH, pH 7.9, 0.1% SDS, 500 mM NaCl, 1% Triton X-100 2 mM EDTA, one time with 100 mM Tris-HCl pH 7.5, 0.5 M LiCl, 1% NP-40, 1% Sodium Deoxycholate and one time with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. The DNA was eluted twice by incubating for 30 minutes in 25 ul of 20 mM HEPES-KOH, pH 7.9, 1 mM EDTA, 0.5% SDS, 0.5 mg/ml Proteinase K. To the 50 ul of eluted DNA, 3 ul of 3M Sodium Acetate, pH 5.3 and 0.5 ul 30 mg/ml RNase A was added and incubated overnight at 65˚C in a hybridization oven. 1.5 ul of 20 mg/ml proteinase K was added and incubated for 1 hour at 50˚C and the DNA was purified with a 2X volume of a homemade version of AMPure XP beads and eluted in 20 ul Tris-HCl, pH 8.0, 0.1 mM EDTA. Libraries were made with the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) according to the manufacturer’s instructions.
identifiers.namespace: GEO
identifiers.id: GSM2735962