title: Replicate 2 Raji iBZLF1 full-length 6h
organism: Homo sapiens
description: Protocols: For Capture-C analysis, 107 uninduced Raji iBZLF1 cells or cells induced for different time periods (6 h, 15 h) with 100 ng/ml doxycycline were used. Cells were washed (PBS), filtered for single cells, and fixed (1% formaldehyde, 10 % fetal bovine serum in PBS). The fixation was quenched on ice with 1 M glycine before cells were homogenized two times with a 20G syringe in 3C lysis buffer (10 mM TrisHCl (pH 7.5), 10 mM NaCl, 0.2 % NP-40 in ddH2O + PIC). Cells were washed and taken up in 1.2x CutSmart buffer (NEB). SDS was added to a concentration of 0.1 % and the cells were incubated (65 °C, 40 min) while shaking (1,200 rpm). Triton X-100 was added to a final concentration of 4 % and the cells were incubated (37 °C, 1 h, 1,200 rpm). The cell suspension was divided into six aliquots of 200 μl each and incubated with 80 units DpnII at 37 °C while shaking (1,200 rpm) overnight followed by another 400 units of DpnII for 4 h. The reaction was stopped with SDS at a final concentration of 0.1 %. Samples were joined and diluted with 1.25x 3C ligation buffer (62.5 mM Tris-HCl (pH 7.5), 12.5 mM MgCl2, 1.25 mM ATP, 12.5 mM DTT in ddH2O) and Triton X-100 (37 °C, 1 h, shaking ever 10 min) to a final volume of 1.5 ml. 100 units T4 DNA Ligase (Affymetrix) was used for DNA fragment ligation (16 °C for 4.5 h and RT for 45 min) followed by proteinase K treatment at 65 °C for 1 h to revert crosslinking. RNAs were degraded with RNase A (300 μg, 37 °C, 1 h). DNA was extracted with organic solvents (2/3 v/v phenol-chloroform and 1/3 v/v butanol), precipitated (EtOH, 100 %), washed (EtOH 70 %), and rehydrated (TE buffer). 1.5 μg DNA was sheared with a Covaris M-series instrument (Covaris, peak incident power: 50 watts; duty factor 20 %; cycles/burst: 200 counts, duration 200 sec). Samples were cleaned with Agencourt AMPure beads (Beckman Coulter). The library preparation and sequence capture were done as described in the SureSelectXT2 Target Enrichment System for Illumina Paired-End Multiplexed Sequencing manual (Agilent, Version: E1, June 2015). The desired fragments were captured with Dynabeads (Thermo Fisher Scientific).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-04-05T04:02:49Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-03-26T23:18:48Z
attributes.tag: External Id
attributes.value: SAMEA5537258
attributes.tag: INSDC center name
attributes.value: Research Unit Gene Vectors Helmholtz Zentrum Munchen German Research Center for Environmental Health and German Center for Infection Research (DZIF), Partner site Munich, Germany
attributes.tag: INSDC first public
attributes.value: 2019-04-05T04:02:49Z
attributes.tag: INSDC last update
attributes.value: 2019-03-26T23:18:48Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7825:Replicate 2 Raji iBZLF1 full-length 6h
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: Raji
attributes.tag: cell type
attributes.value: B cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: Burkitts lymphoma
attributes.tag: genotype
attributes.value: doxycycline-inducible full-length BZLF1
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-7825:Replicate 2 Raji iBZLF1 full-length 6h
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: stimulus
attributes.value: doxycycline