|
| Status |
Public on Apr 16, 2021 |
| Title |
E14_S2 Micro Capture-C (MCC) |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Embryonic stem cells
|
| Treatment protocol |
Cells were resuspended in aliquots of 1 x 107 in 10 ml media with 10% FCS in a 15-ml conical centrifuge tube using an automated cell counter (CountessII, Thermo Fisher). Formaldehyde (37% (vol/vol)) was added to each aliquot to make a final concentration of 2% (vol/vol) and the cells were incubated on a roller mixer for 10 minutes at room temperature. The crosslinking reaction was quenched with cold 1 M glycine to a final concentration 130 mM and the sample was centrifuged (5 min at 220 g). The supernatant was discarded and the cell pellet was washed with 10 ml cold Phosphate Buffered Saline (PBS) and reconstituted in 1 ml PBS. An automated cell count on was performed on 10 ml of the cell suspension to reconfirm the cell number. Digitonin (Sigma D-141) was prepared fresh for each experiment (0.5% in dimethylsulfoxide (DMSO) (wt/vol)). Each batch of digitonin was tested for its ability to permeabilise cells at a concentration of 0.005% (wt/vol). 10 ml digitonin was added to make a final concentration of 0.005% (wt/vol). For standard nuclear preparation, the pellet was resuspended in 5ml cold lysis buffer (TRIS-HCl pH 8 10 mM, NaCl 10 mM, Igepal NP-40 (Sigma I8896) 0.2%, cOmplete proteinase inhibitor cocktail (PIC) (Roche 4693116001) (x1 tablet / 50 ml)) and incubated on ice for 20 min. The nuclei were centrifuged (300 g, 5 min) and resuspended in 1ml lysis buffer.
|
| Growth protocol |
Erythroid cells were obtained from the spleen of mice treated with phenylhydrazine (40mg/g body weight x3 doses 12h apart; sacrificed on day 5). Phenylhydrazine causes haemolytic anaemia and marked erythroid expansion in the spleen so that >80% all cells are CD71+ Ter119+ erythroid precursors. Mouse ES cells (E14) were routinely cultured in Glasgow MEM x1 liquid (Gibco 21710-025) with 10% FCS (vol/vol) (Gibco), 1x Glutamine (Gibco 25030-024), 1 mM sodium pyruvate (Gibco 11360-039), 1x non-essential amino acids (Gibco 11140-035), mercaptoethanol (Gibco 31350010) and LIF (Thermo Fischer PMC9484) in gelatin (Gibco 10131-035) coated flasks. Cells were harvested when 80-95% confluent using trypsin (Sigma T-4049) and washed once prior to fixation. HUDEP-2 cells were cultured in expansion phase as previously described Canver, M. C. et al.,2015.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Qiagen DNeasy blood and tissue kit
7-10 µg of each 3C library was sonicated to a mean fragment size of 200 bp using a Covaris S220 Focussed Ultrasonicator (1-3 cycles of 60s: duty cycle 10%; intensity 5; cycles per burst 200). The library size was assessed using an Agilent TapeStation (DNA 1000) and an Ampure XP (Beckman Coulter) bead clean-up was performed once the sample had been adequately sonicated. To maximise library complexity three aliquots, each containing 2 µg sonicated 3C library, were processed in parallel for each sample to add sequencing adaptors. NEB Ultra II reagents were used following the standard protocol with the following modifications: first, additional adapter was added into the ligation reactions (7.5 µl rather than 2.5 µl); no size selection was performed (Ampure XP bead clean ups were always performed with a sample:bead ratio of 1:1.8) and the final PCR to add indices was amplified in triplicate from each aliquot using the Herculase II PCR kit (Agilent) to maximise DNA yield. All parallel library preparations and PCR reactions were pooled for each reaction. CHIP libraries were prepared and analyzed as previously reported in Hanssen LLP. et al., 2017.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Library strategy: Micro Capture-C (MCC)
The raw fastq file is processed to reconstruct a single read from the paired end sequencing. This is then mapped to the 800bp sequence surrounding the capture oligonucleotide using the non-stringent aligner BLAT. This allows the reads to be cut into ‘slices’ depending on whether they align to the sequence around the capture site. This strategy allows ligation junctions in the read to be determined with base pair accuracy. The resulting fastq file is aligned to the genome using bowtie.
CHIP libraries were prepared and analyzed as previously reported in Hanssen LLP. et al., 2017.
Genome_build: mm9, hg19
Supplementary_files_format_and_content: BigWig of data from multipul viewpoints
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| |
|
| Submission date |
Sep 11, 2020 |
| Last update date |
Apr 16, 2021 |
| Contact name |
Peng Hua |
| E-mail(s) |
penghua@njmu.edu.cn
|
| Organization name |
Nanjing Medical University
|
| Department |
State Key Laboratory of Reproductive Medicine and Offspring Health
|
| Street address |
101 Longmiandadao
|
| City |
Nanjing |
| State/province |
Jiangsu |
| ZIP/Postal code |
211116 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE144336 |
Defining genome architecture at base-pair resolution |
|
| Relations |
| BioSample |
SAMN16112141 |
| SRA |
SRX9107985 |
