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Status |
Public on Jun 07, 2021 |
Title |
E14_RFPneg_CTCF |
Sample type |
SRA |
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Source name |
mouse ESC_E14
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Organism |
Mus musculus |
Characteristics |
cell type: ESC cell line: E14 genotype: wild type treatment: None antibody: anti-CTCF Antibody (Millipore, Cat# 07-729, RRID:AB_441965)
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Treatment protocol |
Doxycyline was added for 16h or 24h to the media when indicated
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Growth protocol |
Wild-type (R1 and E14) ESC and ESCDux were grown on a feeder layer of growth-arrested MEFs or on gelatin 0.1% in high-glucose DMEM (Invitrogen) supplemented with 15% FBS, 1:500 LIF (made in house), 0.1 mM nonessential amino acids, 1% glutamax, 1mM Sodium Pyruvate, 55 mM β-mercaptoethanol, and 1% penicillin/streptomycin (all from Life Technologies) at 37°C and 5% CO2. Cells were routinely passaged with Trypsin 0.05% (Gibco).
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Extracted molecule |
genomic DNA |
Extraction protocol |
The CUT&RUN protocol was slightly modified from (Skene et al., 2018; Meers et al., 2019). In brief, trypsinized or cell sorted ESC (between 150,000-500,000 cells depending on the experiment) were washed three times with Wash Buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, Roche complete Protease Inhibitor tablet EDTA free) and bound to activated Concanavalin A beads (Polysciences) for 10 minutes at room temperature. Cells were then permeabilized in Digitonin Buffer (0.05 % Digitonin and 0.1% BSA in Wash Buffer) and incubated with the antibody against CTCF (07-729, Millipore) at 4°C for 2 hours. For negative controls, Guinea Pig anti-Rabbit IgG (ABIN101961, Antibodies-online) was used. Cells were washed with Digitonin Buffer following antibody incubation, and further incubated with purified hybrid protein A-protein G-Micrococcal nuclease (pAG-MNase) at 4°C for 1 hour. Samples were washed in Digitonin Buffer, resuspended in 150 μl Digitonin Buffer and equilibrated to 0°C on ice water for 5 minutes. To initiate MNase cleavage, 3 μl 100 mM CaCl2 was added to cells and after 1 hour of digestion, reactions were stopped with the addition of 150 μl 2x Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02 % Digitonin, 50 μg/ml RNase A, 50 μg/ml Glycogen). Samples were incubated at 37°C for 10 minutes to release DNA fragments and centrifuged at 16,000 g for 5 minutes. Supernatants were collected and a mix of 1.5 μl 20% SDS / 2.25 μl 20 mg/ml Proteinase K was added to each sample and incubated at 65°C for 35 minutes. DNA was precipitated with ethanol and sodium acetate and pelleted by high-speed centrifugation at 4°C, washed, air-dried and resuspended in 10 μ 0.1x TE. The entire precipitated DNA obtained from CUT&RUN was used to prepare Illumina compatible sequencing libraries. In brief, end-repair was performed in 50 μl of T4 ligase reaction buffer, 0.4 mM dNTPs, 3 U of T4 DNA polymerase (NEB), 9 U of T4 Polynucleotide Kinase (NEB) and 1 U of Klenow fragment (NEB) at 20°C for 30 minutes. End-repair reaction was cleaned using AMPure XP beads (Beckman Coulter) and eluted in 16.5 μl of Elution Buffer (10 mM Tris-HCl pH 8.5) followed by A-tailing reaction in 20 μl of dA-Tailing reaction buffer (NEB) with 2.5 U of Klenow fragment exo- (NEB) at 37°C for 30 minutes. The 20 μl of the A-tailing reaction were mixed with Quick Ligase buffer 2X (NEB), 3000 U of Quick Ligase (NEB) and 10 nM of annealed adaptor (Illumina truncated adaptor) in a volume of 50 μl and incubated at room temperature for 20 min. The adaptor was prepared by annealing the following HPLC-purified oligos: 5′-Phos/GATCGGAAGAGCACACGTCT-3′ and 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC∗T-3′ (∗phosphorothioate bond). Ligation was stopped by adding 50 mM of EDTA, cleaned with AMPure XP beads and eluted in 14 μl of Elution Buffer. All volume was used for PCR amplification in a 50 μl reaction with 1 μM primers TruSeq barcoded primer p7, 5′-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC∗T-3′ and TruSeq barcoded primer p5 5′-AATGATACGGCGACCACCGAGATCTACACXXXXXXXXACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ (∗ represents a phosphothiorate bond and XXXXXXXX a barcode index sequence), and 2X Kapa HiFi HotStart Ready mix (Kapa Biosciences). The temperature settings during the PCR amplification were 45 s at 98°C followed by 15 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C and a final 5 min extension at 72°C. PCR reactions were cleaned with AMPure XP beads (Beckman Coulter), run on a 2% agarose gel and a band of 300bp approximately was cut and gel purified using QIAquick Gel Extraction Kit (QIAGEN). Library concentration was determined with KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems). Sequencing was performed on the Illumina NextSeq550 (75bp pair-end reads).
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
NextSeq 550 |
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Data processing |
Library strategy: CUT&RUN Basecalling was performed using bcl2fastq version 2.20.0 Data were processed using a modified version of Cut&RunTools (Zhu et al., 2019). Reads were adapter trimmed using fastp v.0.20.0 (Chen et al., 2018). An additional trimming step was performed to remove up to 6bp adapter from each read. Next, reads were aligned to a hybrid mm10 and E. coli (MG1655) genome using bowtie2 (Langmead et al., 2012) with the ‘dovetail’ and ‘sensitive’ settings enabled. Aligned fragments were segregated into mouse and carry-over (E. coli) subsets. macs2 (Zhang et al., 2008) was used to call peaks with q-value cutoff < 0.01. Normalized reads per kilobase per million (RPKM) signal tracks were generated using the ‘bamCoverage’ utility from deepTools (Ramirez et al., 2016) with parameters ‘center_reads’ and ‘extend_reads’ options enabled. A bin-size of 25bp and smooth length of 75bp was used for Nanog, Otx2, and Sox2 data, and bin-size 75 and smooth-length 300bp for H3K27ac data. Genome_build: mm10 Supplementary_files_format_and_content: bigwig files containing RPKM normalized read density.
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Submission date |
Jan 20, 2021 |
Last update date |
Jun 08, 2021 |
Contact name |
Desiree Tillo |
E-mail(s) |
desiree.tillo@nih.gov
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Phone |
+1-240-760-7289
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Organization name |
NIH/NCI
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Street address |
41 Center Dr, Room D310
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City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platform ID |
GPL21626 |
Series (2) |
GSE165160 |
CTCF is a Barrier for Totipotent-like Reprogramming [Cut&Run] |
GSE165162 |
CTCF is a Barrier for Totipotent-like Reprogramming |
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Relations |
BioSample |
SAMN17387479 |
SRA |
SRX9901787 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5027357_E14_RFPneg_CTCF.mm10.RPKM.bw |
117.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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