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| Status |
Public on Feb 06, 2024 |
| Title |
BG4_MUT_Bio3.Tech1 |
| Sample type |
SRA |
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| Source name |
HEK293T_MutMYCG4
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T_MutMYCG4
cell type: Human embryonic kidney
chip antibody: BG4
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| Growth protocol |
Human embryonic kidney (HEK293T), cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco, 41966-029) growth media supplemented with 10% fetal bovine serum (Gibco, A3840401) and 2 mM L-Glutamine (Gibco, 25030024). Cells were maintained at standard conditions (5% CO2, 37°C) on TC-treated 6-well plates (Corning, 3516) and regularly tested for mycoplasma contamination (RICS, CRUK-CI Core Facility). For passaging, cells were washed with DPBS pH 7.4 (Gibco, 14190094), detached with StemPro Accutase (Gibco, A11105-01) at 37°C for 5mins and resuspended in 10-fold excess of growth media. Wild type J1 (129/Sv/Jae) mouse embryonic stem cell (mESC) cultures were routinely maintained in DMEM high glucose (Sigma, D6546) containing 10% FBS (Gibco, 16141079), 1x GlutaMax-I (Gibco, 35050-038), 1x NEAA (Gibco, 11140-035), 0.1 mM β-mercaptoethanol (Sigma, M3148 diluted to 50 mM in 50 µM EDTA/PBS solution), 1000 U/mL recombinant LIF (PeproTech, murine LIF, 250-02) and 1 µM PD (Sigma, PZ0162-5MG), 3 µM CHIR (Cambridge biosciences, HY-10182A-5MG). Cells were cultured under standard conditions at 37°C in water saturated, CO2 (5%) enriched atmosphere on 0.2% (w/v) gelatine-coated (Sigma, G9391) plates and regularly tested for mycoplasma contamination (RICS, CRUK CI core). Authentication of mESCs was performed by mouse STR analysis (RICS, CRUK CI core). For passaging cells were washed with PBS (Gibco, 14190094), detached with TrypLE (Gibco, 12604-013) at 37°C for 5 min and resuspended in 10-fold excess of mESC growth medium as described.
Human embryonic kidney (HEK293T), cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco, 41966-029) growth media supplemented with 10% fetal bovine serum (Gibco, A3840401) and 2 mM L-Glutamine (Gibco, 25030024). Cells were maintained at standard conditions (5% CO2, 37°C) on TC-treated 6-well plates (Corning, 3516) and regularly tested for mycoplasma contamination (RICS, CRUK-CI Core Facility). For passaging, cells were washed with DPBS pH 7.4 (Gibco, 14190094), detached with StemPro Accutase (Gibco, A11105-01) at 37°C for 5mins and resuspended in 10-fold excess of growth media. Wild type J1 (129/Sv/Jae) mouse embryonic stem cell (mESC) cultures were routinely maintained in DMEM high glucose (Sigma, D6546) containing 10% FBS (Gibco, 16141079), 1x GlutaMax-I (Gibco, 35050-038), 1x NEAA (Gibco, 11140-035), 0.1 mM β-mercaptoethanol (Sigma, M3148 diluted to 50 mM in 50 µM EDTA/PBS solution), 1000 U/mL recombinant LIF (PeproTech, murine LIF, 250-02) and 1 µM PD (Sigma, PZ0162-5MG), 3 µM CHIR (Cambridge biosciences, HY-10182A-5MG). Cells were cultured under standard conditions at 37°C in water saturated, CO2 (5%) enriched atmosphere on 0.2% (w/v) gelatine-coated (Sigma, G9391) plates and regularly tested for mycoplasma contamination (RICS, CRUK CI core). Authentication of mESCs was performed by mouse STR analysis (RICS, CRUK CI core). For passaging cells were washed with PBS (Gibco, 14190094), detached with TrypLE (Gibco, 12604-013) at 37°C for 5 min and resuspended in 10-fold excess of mESC growth medium as described.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&Tag and Chem-map were performed as previously described (Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019),Yu, Z., et al. Nat Biotechnol (2023)).G4 mapping (BG4, SG4, SG4mut-R105A) and R-loop profiling were performed as 3-layer CUT&Tag on DynabeadsTMMyOneTMStreptavidinT1 (Thermo Fisher, cat#65601). SG4 and SG4mutR105A CUT&Tag was performed in extracted nuclei as previously described (Hui W.W.I., et al. Sci Rep (2021)). PDS Chem-map, G4 interactome CUT&Tag experiments (SP1, CNBP, MLL1, MLL4, SIN3A, SMARCA4, RNAPIIS5P, RNAPIIS2P) and histone marks (H3K4me1, H3K4me3, H3K27me) were performed as 2-layer CUT&Tag on ConcanavalinA (ConA)-coated magnetic beads (Bangs Laboratories, BP531).MyOneT1 beads were washed twice with PBS pH 6.8, resuspended in 1xPBS pH 6.8 supplemented with 0.01% Tween-20 and mixed with the biotin-conjugated ConA (Sigma-Aldrich, cat#C5275-5MG) solution at 2.3mg/mL at a 1:0.5 ratio. The mix was then incubated at 22°C at 400 rpm for 30min. Aliquots of 500,000 cells/100mL were added to 10mL of beads, washed twice with 100mL wash buffer and resuspended in 1% BSA Antibody Buffer. Primary antibodies were added (1:50), and the mix was incubated at 4°C overnight at 600 rpm. Cells were then washed twice with 100mL dig-wash buffer (0.05% digitonin, 20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and resuspended in 50mL dig-wash buffer. Secondary antibodies were added 1:50 to the samples and incubated at 25°C for 2h at 600 rpm (2-layer CUT&Tag). For G4-mapping samples, cells were washed three times with 500mL dig-wash buffer and resuspended in 50mL dig-wash buffer and 0.5mL of guinea-pig anti-rabbit antibody (Antibodies online, ABIN101961)was added and incubated with the cells at 25°C for 1h at 600 rpm (3-layer CUT&Tag). Cells were then washed three times with 100mL Dig-300 buffer (0.01% digitonin, 20mM HEPES pH7.5, 300mM KCl, 0.5mM spermidine in nuclease-free water with a Roche Complete Protease Inhibitor EDTA-free tablet) and incubated in 1:250 dilution of pA-Tn5 in 50µL Dig-300 buffer at 25°C for 1h at 600rpm.Cells were then washed three times with 500 µL Dig-300 buffer and the Tn5 tagmentation was activated by incubating in 300mL Dig-300 buffer supplemented with 10 mM MgCl2at 37°C for 1h at 600 rpm. Cells were then washed twice with 500 µL TAPS buffer and resuspended in 0.5 mg/mL Proteinase K, 0.5% SDS in 10 mM Tris-HCl buffer pH 8.0 at 55°C for 1h at 800 rpm. Tagmented DNA was purified with DNA clean & concentrator columns (Zymo Research, D4013).
For Illumina library preparation, genomic DNA extracted by CUT&Tag was used. Libraries were generated as previously described with a universal i5 and a uniquely barcoded i7 primer(Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. Nat Commun 10, 1930 (2019)). Libraries were visualized and quantified on a high-sensitivity D1000 tape on a Tape Station (AgilentTechnologies 2200)andwith the NEBNext®Library Quant Kit for Illumina (NEB, cat#7630S), following the manufacturer’s instructions.Libraries were sequenced on an Illumina NextSeq 500 (High Output Kit v2.5, 75 cycles) or NextSeq 2000 instrument(P2 or P3 reagents, 100 cycles). RNA-seq and library preparation Total RNA was extracted from 1.0x106 cells using the RNeasy Plus Mini Kit (Qiagen, cat#74134) as per manufacturer’s instructions. RNA quality was checked via TapeStation (Agilent Technologies 2200, RIN>9) and RNA concentration determined using a Qubit fluorometer (Thermo Fisher, Qubit 4). Libraries were generated from 700ng RNA using the NEBNext®Poly(A) mRNA Magnetic Isolation Module (NEB, cat#7490S) together with the NEBNext®UltraTMII Directional RNA Library Prep Kit for Illumina (NEB, cat#7760S) as per manufacturer’s instructions. Libraries were sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles). For MNase 1.0 x 106 cells were washed in ice-cold PBS and incubated in 1mL lysis buffer (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT, 0.1% NP-40) for 10 mins on ice. Cells were then centrifuged at 1,000 x g for 5 mins at 4 °C and resuspended in lysis buffer without NP-40 (10 mM Tris-HCl pH 8.0, 60 mM KCl, 15 mM NaCl, 3 mM MgCl2, 1 mM DTT). Cells were then incubated at 37 °C for 5 mins supplemented with 2 mM CaCl2 and digested with 10 U MNase at 37 °C for 60 mins. The digestion was stopped with a final concentration of 40 mM EDTA, 0.5% SDS. DNA was incubated with RNase A (Thermo Scientific, cat #EN0531) for 1 h at 37oC. DNA was purified using Phenol-chloroform-isoamyl alcohol (PCl, Thermo Scientific, cat #15593049). Libraries were generated with the NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, cat #7760S) as per manufacturer’s instructions. A bead-based size selection of 200 bp was performed to isolate mononucleosomal fragments. Libraries were then quantified with the NEBNext® Library Quant Kit for Illumina (NEB, cat #7630S) and sequenced on a NextSeq 500 system (High Output Kit v2.5, 75 cycles).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
fq files were trimed with cutadapt version 1.18 to remove adaptors and bases with quality lower than 20 (-q 20) mapped with bwa-mem 0.7.17-r2188 to hg38 genome and the coresponding modified genomes, and only the reads within the whitelist have been retained.
bw files presented here were created using bamCoverage with counts per milion normalisation (CPM) after extending the paired-end reads and accepting maximum mapped fragment length of 350nt. For the polymerase tracks the tracks were further rescaled, normalised for the E coli reads in the library.
Assembly: hg38 , hg38_MutMYCG4, hg38_SwapKRASG4, hg38_MYCFlip_StrandSwap, mm10
Supplementary files format and content: bigWig
Library strategy: CUT&Tag
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| Submission date |
Sep 30, 2023 |
| Last update date |
Feb 06, 2024 |
| Contact name |
Larry Melidis |
| E-mail(s) |
lm512@cam.ac.uk
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| Organization name |
University of Cambridge
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| Department |
CRUK-CI
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| Lab |
Balasubramanian
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| Street address |
Robinson Way
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| City |
Cambridge |
| State/province |
Not US or Canada |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE223370 |
G-quadruplex DNA structure is a positive regulator of MYC transcription |
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| Relations |
| BioSample |
SAMN37628444 |
| SRA |
SRX21954909 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7815654_BG4_MUT_Bio3.Tech1.merged.s350.cpm.bs3.bw |
221.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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