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Sample GSM6244116

Query DataSets for GSM6244116

Status

Public on Jun 18, 2022

Title

Set2 K562_Micro-C Replicate B

Sample type

SRA

Source name

K562

Organism

Homo sapiens

Characteristics

cell line: K562

Treatment protocol

For the PARP1 inhibition experiment, cells were treated with either 10 μM Olaparib, initially diluted in DMSO, or DMSO only, for 90 min before immediately crosslinked for Micro-C or lysed in NUN buffer (0.3 M NaCl, 1 M Urea, 1% NP-40, 20 mM HEPES, pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 20 units per ml SUPERase In Rnase Inhibitor (Life Technologies, AM2694), 1X Protease Inhibitor Cocktail (Roche, 11 873 580 001)) for ChRO-seq. To induce NELFB degradation, dTAG-13 (Bio-Techne) was reconstituted in DMSO (Sigma) at 5 mM. dTAG-13 was diluted in maintenance medium to 500 nM and added to cells with medium changes for the specified amounts of time. For dTAG washes, the cells were washed 4 times, twice with PBS +/+ and twice with maintenance medium following the treatment time to ensure complete removal of the dTAG ligand. At the end of each dTAG-13 treatment time point, cells were detached using Trypsin-EDTA (0.05%) (Gibco) and counted before crosslinking for Micro-C.

Growth protocol

K562 and Jurkat cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1X penicillin streptomycin antibiotic. mECSs harboring a homozygous endogenous NELFB-FKBP12F36V fusion protein were cultured on 0.1% gelatin (Millipore) in PBS+/+ coated tissue-culture grade plates in a humidified 37°C incubator with 5% CO2. For routine culture, cells were grown in Serum/LIF conditions: DMEM (Gibco), supplemented with 2 mM L-glutamine (Gibco), 1x MEM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 15% Fetal Bovine Serum (Gibco), and 1000 U/ml of recombinant leukemia inhibitory factor (LIF).

Extracted molecule

genomic DNA

Extraction protocol

Cells were crosslinked with 1 ml per million cells of 1% formaldehyde for 10 minutes at room temperature and quenched by 0.25 M Glycine for 5 min. After spin-down for 5 minutes at 300Xg at 4 °C, cells were washed at a density of 1 ml per million cells in ice cold PBS. Cells were crosslinked a second time, with 1 ml per 4 million cells of 3 mM disuccinimidyl glutarate (DSG) (ThermoFisher Scientific, 20593) for 40 min at room temperature and quenched by 0.4 M Glycine for 5 min. Following two washes with ice cold PBS, cells were flash-frozen and kept at -80 °C until further use. For MNase digestion, cells were thawed on ice for 5 min, incubated with 1ml MB#1 buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1x Roche cOmplete EDTA-free (Roche diagnostics, 04693132001)) and washed twice with MB#1 buffer. MNase concentration for each cell type was predetermined using MNase titration experiments exploring 2.5-20U of MNase per million cells. We selected the concentration that gives ~90% mononucleosomes in an MNase. Chromatin was digested with MNase for 10 min at 37 °C and digestion was stopped by adding 8 ul of 500 mM EGTA and incubating at 65 °C for 10 min. Following dephosphorylation with rSAP (NEB #M0371) and end polishing using T4 PNK (NEB #M0201), DNA polymerase Klenow fragment (NEB #M0210) and biotinylated dATP and dCTP (Jena Bioscience #NU-835-BIO14-S and #NU-809-BIOX-S, respectively), ligation was performed in a final volume of 2.5 ml for 3h at room temperature using T4 DNA ligase (NEB #M0202). Dangling ends were removed by a 5 min incubation with Exonuclease III (NEB #0206) at 37 °C and biotin enrichment was done using 20 ul DynabeadsTM MyOneTM Streptavidin C1 beads (Invitrogen #65001).
Libraries were prepared with the NEBNext Ultra II Library Preparation Kit (NEB #E7103). Samples were sequenced on a combination of Illumina’s NovaSeq 6000 and HiSeq 2500 at Novogene.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

Micro-C
K562.mapq_30.mcool
K562_cis_mapq30_pairs.gz

Data processing

All Micro-C mapping was done using the mirnylab/distiller-nf: v0.3.3 pipeline. Raw data were mapped to the hg38 human genome assembly (K562 and Jurkat) or mm10 mouse genome assembly (mESCs).
For data visualization by contact maps, multi cool (mcool) files, balanced by iterative correction and eigenvector decomposition (ICE) for resolutions of 200 bp to 10 Mb were generated from contacts with both ends having a mapq score > 30.
Assembly: hg38, mm10
Supplementary files format and content: pairs file - contains the coordinated of read pair ends, mapq and orientation.
Supplementary files format and content: mcool file - contains all valid Micro-C pairs stored in HDF5 format and ICE-normalized with mutiple resolutions
Library strategy: Micro-C

Submission date

Jun 14, 2022

Last update date

Jul 21, 2022

Contact name

Charles Grahe Danko

E-mail(s)

dankoc@gmail.com

Organization name

Cornell University

Department

Baker Institute for Animal Health