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| Status |
Public on Jun 05, 2018 |
| Title |
HeLa_mCherry_control_sorted_bsPCR-seq_rep2 |
| Sample type |
SRA |
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| Source name |
HeLa
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| Organism |
Homo sapiens |
| Characteristics |
cell type: human cervical cancer cell line
plasmids: pEf1a-mCherry-T2A-Puro
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| Treatment protocol |
Cells were seeded 24h prior to transfection. Cells were transfected transiently with plasmids containing the guide RNA, dC9Sun-D3A, dC9-D3A, mutant or mCherry controls. 48h post-transfection, cells were puromycin (2 ug/mL, Life technologies, Cat no: A1113803) for 48h. Cells were then harvested and subjected for DNA extraction for bsPCR-seq or cross-linked for ChIP-seq and ChIP-bs-seq experiments.
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| Growth protocol |
HeLa cells were cultured with DMEM (Dulbecco's Modified Eagle Medium, Life Tecnologies) supplemented with 10% (v/v) FBS (Life Technologies) and 1X (v/v) Glutamax (Life Technologies)
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| Extracted molecule |
genomic DNA |
| Extraction 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.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina MiSeq |
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| Description |
targeted PCR amplicons of bisulfite treated genomic DNA (bsPCR-seq)
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| Data processing |
Whole Genome Bisulfite Sequencing by MethylC-seq. Reads were mapped to the human genome (hg19) with the Bowtie alignment algorithm (Langmead et al. 2009) with the following parameters: -e 1120 –O 20 –n 1 as previously reported (Lister et al. 2011).
ChIP-seq and ChIP-bisulfite seq. Reads were trimmed with Trimmomatic (Bolger et al. 2014) with the following parameters: ILLUMINACLIP:2:30:10, LEADING:3, TRAILING:3, SLIDINGWINDOW:4:20, MINLEN:25. For ChIP-seq reads were mapped with the bowtie software package (Langmead et al. 2009) to the human genome (hg19) and non-uniquely mapped reads were discarded. For ChIP-bisulfite-seq reads were 10 bp hard trimmed on the 3' end (Accel-NGS Methyl-Seq DNA library kit requirement), mapped and methylation was called as using BS-Seeker2 (Guo et al. 2013) with default parameters (Bowtie 1), allowing for one mismatch.
bsPCR-seq. Reads were mapped and methylation was called as using BS-Seeker2 (Guo et al. 2013) with default parameters (Bowtie 1), allowing for one mismatch.
Genome_build: hg19
Supplementary_files_format_and_content: For the WGBS data the 'allC' file format is a tab-delimited text file with the following columns: 1) chromosome, 2) position, 3) strand, 4) context, 5) cytosine base calls, and 6) all base calls. For the ChIP-BS-seq data the 'allC' file format is a tab-delimited text file with the following columns: 1) chromosome, 2) position, 3) position, 4) strand, 5) context, 6) cytosine base calls, and 7) all base calls. For the bsPCR-seq data the 'allC' file format is a tab-delimited text file with the following columns: 1) chromosome, 2) position, 3) position, 4) strand, 5) context, 6) cytosine base calls, and 7) all base calls.
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| Submission date |
Dec 01, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Christian Pflueger |
| E-mail(s) |
christian.pflueger@uwa.edu.au
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| Organization name |
University of Western Australia
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| Department |
Harry Perkins Institute of Medical Research
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| Lab |
Ryan Lister Lab
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| Street address |
6 Verdun Street
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| City |
Nedlands |
| State/province |
Western Australia |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE107607 |
A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs |
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| Relations |
| BioSample |
SAMN08120146 |
| SRA |
SRX3437489 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2872372_dCas9_sorted_Control_R2.allC.txt.gz |
20.9 Kb |
(ftp)(http) |
TXT |
| GSM2872372_dCas9_sorted_Control_R2.sym.CG.bigwig |
21.9 Kb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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