|
| Status |
Public on Sep 13, 2016 |
| Title |
DSG_pellet-1 |
| Sample type |
SRA |
| |
|
| Source name |
Yeast
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
media: YPD
first fixation: 3% Formaldehyde for 10 min
second fixation: 3mM DSG for 40min
|
| Treatment protocol |
1) Protocol for formaldehyde only: Yeast cells were fixed with 3% formaldehyde for 15 min and quenched with 125 mM glycine for 5 min. Cells were pelleted, spheroplasted with Zymolyase, and treated with a level of MNase yielding >95% mononucleosomes. 2) Protocol for long crosslinking: Yeast cells were fixed with 3% formaldehyde for 10 min, and quenched with 125 mM glycine for 5 min. Cells were pelleted and spheroplasted with Zymolyase. The spheroplasts were washed by PBS, subjected to long-crosslinker fixation for 40min, and quenched with 250 mM glycine for 5 min. Cells were pelleted again, washed with PBS, and treated with a level of MNase yielding >95% mononucleosomes. After stopping MNase, chromatin pellet and supernatant was dephosphorylated using rSAP separately. Crosslinked chromatin was subject to T4 DNA polymerase / T4 PNK enzyme mix solution to generate blunt ends. Chromatin was diluted into 2.5 mL and treated with T4 DNA ligase. Ligated products were treated with 100U of exonuclease III for 5 min to eliminate biotinylated ends of unligated DNA. Proteinase K was then added and incubated at 65 C overnight.
|
| Growth protocol |
The 100mL yeast cultures were grown to midlog phase ~OD 0.55 in indicated media at 30 C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was purified by PCI extraction and ethanol precipitation, treated with RNase A, and ~250-350 bp DNA was gel-purified following 3% agarose gel electrophoresis.
Purified DNA was treated with End-it, subject to A-tailing with Exo- Klenow, and ligated to Illumina adaptors. Adaptor-ligated DNA was then purified with streptavidin beads to isolate ligated Micro-C products away from undigested dinucleosomal DNA. Streptavidin beads were then subject to ~10-13 cycles of PCR using Illumina paired-end primers.
Amplified library was purified and subject to Illumina Nextseq 500 paired end sequencing with 40 x 40 bp reads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: Micro-C XL
OLB 1.9/CASAVA_v1.8.2 were used for base calling
Barcoded libraries were demultiplexed by Novobarcode.
Paired reads were mapped to reference genome separately by Bowtie2
Parse SAM files and remove duplicate reads.
Mate unique paired mates and generate contact matrix.
Genome_build: S288C (sacCer3) or S. pombe 972h- (ASM294v2)
Supplementary_files_format_and_content: Interaction_density.xlsx
|
| |
|
| Submission date |
Aug 04, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Xavier Darzacq |
| E-mail(s) |
darzacq@berkeley.edu
|
| Phone |
510-642-0884
|
| Organization name |
University of California, Berkeley
|
| Department |
Molecular and Cell Biology
|
| Lab |
Darzacq Lab
|
| Street address |
475D Li Ka Shing Center
|
| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE85220 |
Micro-C XL: assaying chromosome conformation at length scales from the nucleosome to the entire genome |
|
| Relations |
| BioSample |
SAMN05513377 |
| SRA |
SRX2000986 |
