alias: GSM2872454
title: GSM2872454: pLVP_dCas9_DNMT3A_highActivity_Mir152_3_sgRNA_rep2; Homo sapiens; Bisulfite-Seq
library_construction_protocol: DNA extraction was performed with ISOLATE II Genomic DNA Kit (BIO-52067) according to manufacturer's instructions Whole Genome Bisulfite Sequencing by MethylC-seq libraries were constructed as follows. Genomic DNA was extracted from cells using ISOLATE II genomic DNA extraction kit (Bioline, Cat no: BIO-52067) 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). 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 0.5-1 × 10∧8 cells/ml. Cells are then sonicated to a fragment size range of 500–800 bp. Samples were then diluted in 1.7 ml low-salt buffer and incubated with 5 ug of anti-CTCF (Cell Signaling, 2899B) or anti-NRF1 antibody (Abcam, ab55744), respectively conjugated to a 50:50 mix of protein A/G Dynabeads (Invitrogen, M-280, 20 ul each). After wash steps, DNA was eluted, crosslinks were reversed, and immunoprecipitated DNA was purified by Agencourt AMPure XP beads (Beckman Coulter, Cat no: A63880). Libraries were prepared from the entire ChIP eluate volume containing either 1 ng or 0.1 ng input material per replicate for CTCF or NRF1, respectively, using the ThruPLEX DNA-seq 12S kit (Rubicon Genomics, R400429). After limited PCR amplification, libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Cat no: A63880). ChIP-bisulfite sequencing 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 500 x g 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 microTUBE. The chromatin was sheared with a Covaris S220 sonicator with the following settings: Time 5 min, duty cycle 5%, intensity 6, 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: GSM2872454
attributes.tag: GEO Accession
attributes.value: GSM2872454