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| Status |
Public on Feb 16, 2019 |
| Title |
Leu3_saturated_input_1 |
| Sample type |
SRA |
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| Source name |
TAP-tagged Leu3
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
growth conditions: saturated in YPD
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| Treatment protocol |
Cells were crosslinked by addition of 37% formaldehyde to 1% v/v and incubation at room temperature for 15 minutes, then quenched by addition of glycine to 150 mM and washed with Tris-buffered saline.
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| Growth protocol |
Cells were grown overnight, shaking at 30C in YPD media (1% yeast extract, 2% peptone, 2% dextrose).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinked cell pellets were resuspended in 500 µl FA lysis buffer with 1x Pierce EDTA-free protease inhibitor cocktail, and then 700 µl of 500 µm acid-washed glass beads were added. The cells were vortexed for 12 minutes total, in cycles of 2 minutes shaking and 2 minutes resting on ice. The lysate was pelleted and resuspended in fresh lysis buffer, and then sonicated for 3x10 min runs on a Diagenode Bioruptor, on high power with cycles of 30 seconds on and 30 seconds off, to an average of ~300 bp. The sonicate was cleared by centrifugation, and the resulting supernatant was split into an input aliquot (1/20 of IP volume) and an IP aliquot. For each sample, 10 ul of Pan-Mouse IgG beads (Thermo Fisher Scientific catalog #11041) were washed in fresh lysis buffer for 2 hours and then added to the lysate and then incubated overnight. After washing the beads twice in FA lysis buffer, once in lysis buffer with 500 mM NaCl, twice in RIPA buffer, and once in TE, and the DNA was eluted in 100 ul TE+1% SDS and a second time in 150 ul TE+0.67% SDS. The eluate and inputs were treated with RNase A and Proteinase K and then reverse crosslinked overnight at 65C. DNA was purified using Zymo ChIP DNA Clean & Concentrator and eluted in 15 µl 10 mM Tris-HCl pH 8.
ChIP-seq libraries were prepared using the Accel-NGS 2S Plus DNA Library Kit (Swift) with dual indexing, from either 1 ng total from input samples or 10 ul of IP samples. Input and IP libraries were amplified for 9 and 12-15 cycles, respectively.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Leu3_saturated_merged_FE.bdg.gz
Leu3_saturated_merged_peaks.narrowPeak.gz
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| Data processing |
Basecalls were converted to FASTQ using bcl2fastq 2.17
Sequencing reads were quality-trimmed (cutadapt -q 20), trimmed of adapter sequences, excluding read pairs in which either read was shorter than 28 bp after trimming.
Reads were mapped using bowtie2 2.2.3 using the parameter set --very-sensitive -X 2000
PCR duplicates (with identical fragment start and end positions) were removed.
Coverage across the genome was calculated using bedtools genomecov, using only read pairs with a minimum MAPQ score of 30.
Fold enrichment and significant peaks were calculated for merged replicates using MACS2
genome build: sacCer3
Supplementary_files_format_and_content: for each sample, a bedgraph file of fragment coverage across the genome; for set of replicates, a bedgraph of merged fold enrichment across the genome and a set of called significant peaks (merged files are available in tar archives on the series record).
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| Submission date |
Feb 15, 2019 |
| Last update date |
Feb 17, 2019 |
| Contact name |
Seungsoo Kim |
| Organization name |
Stanford University
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| Department |
Chemical and Systems Biology
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| Lab |
Joanna Wysocka
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| Street address |
265 Campus Dr
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE118118 |
A combination of transcription factors mediates inducible interchromosomal contacts |
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| Relations |
| BioSample |
SAMN10955766 |
| SRA |
SRX5383667 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3610411_Leu3_saturated_input_1.bedgraph.gz |
24.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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