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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 11, 2023 |
| Title |
H3K27ac_B6_HFD_1 [F_B6_1_1] |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
diet: HFD
tissue: Liver
replicate: 1
chip antibody: H3K27ac (Abcam, #4729)
mouse id: F_B6_1_1
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen tissues were Dounce homogenized with loose pestle A in 1× PBS with 1× protease inhibitors (cOmplete, Roche). Tissue suspension was centrifuged at 2000 rpm for 5 min and the pellet was resuspended in 1 ml PBS containing 1 % formaldehyde for 10 min and quenched with glycine 150 mmol/L for 5 min at room temperature. Samples were washed twice with ice-cold 1× PBS with 1× protease inhibitors. Cells were lysed by pelleting and diluting twice in ice-cold SDS lysis buffer (50 mM Tris/HCl, 0.5% SDS, and 10 mM EDTA, 1 × protease inhibitor) and incubating for 10 min at 4°C with gentle mixing. The cell lysate was then Dounce homogenized with tight pestle B in 1 ml ice-cold of SDS lysis buffer and the homogenate was filtered through a 100 µm cell strainer. Samples were split into 3 × 300 µl aliquots in 1.5 sonication tubes and incubated on ice for 10 min. Chromatin was sheared with Bioruptor Plus sonicator (Diagenode) for 35 cycles at high setting. Once cycle was 30s ON, 30s OFF and after each 10 cycles the sonicator was allowed to cool down for 5 min. To neutralize SDS, Triton-X-100 was added to final concentration of 1% along with 50× protease inhibitors (final 1×). All the aliquots were combined and centrifuged at 13,000 rpm for 20 min. Supernatant with sheared chromatin was collected.
ChIP and library preparation were performed using the recently described ChIPmentation protocol (Gustafsson C et al. BMC Genomics. 2019;20(1):59) with minor modifications. For ChIP, 2 µg of rabbit polyclonal anti-CTCF antibody, 5µl of rabbit monoclonal anti-TCF4/TCF7L2 antibody or 2 µg of Anti-Histone H3 (acetyl K27) antibody was added to 50 µl Protein G-coupled Dynabeads (Thermo Fisher Scientific) in 1 × PBS with 0.5% bovine serum albumin (BSA) and rotated at 40 rpm for 4 h at 4°C. Dynabeads were washed three times with PBS with 0.5% BSA and then mixed with 190 µl chromatin samples in 1.5 ml tubes. The samples were rotated at 40 rpm overnight at 4°C. Chromatin was washed with 150 µl of low-salt buffer (50 mM Tris/HCl, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 mM EDTA), high-salt buffer (50 mM Tris/HCl, 500 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 mM EDTA) and LiCl buffer (10 mM Tris/HCl, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and 1 mM EDTA), followed by two washes with TE buffer (10 mM Tris/HCl and 1 mM EDTA) and two washes with ice-cold Tris/HCl pH 8. Chromatin was resuspended in 30 µl of 2 × TD buffer and 1 µl of transposase (Nextera, Illumina) for tagmentation. Samples were incubated at 37°C for 10 min followed by two washes with low-salt buffer. Bead bound tagmented chromatin was diluted in 23 µl of water and 25 µl of 2 × Ultra II Q5 Master Mix (New England BioLabs, M0544S) and 1 µl of both amplification primers (Buenrostro et al, 2013; 0.125 µM final concentration) were added. The libraries were incubated as follows: 72 °C 5 min (adapter extension); 95 °C 5 min (reverse cross-linking); followed by 11 cycles of 98 °C 10s, 63 °C 30s and 72 °C 3 min. After PCR amplification, double-sided purification was performed using SPRIselect beads (Beckman Coulter). To prepare input controls, 2 μl of 50 mM MgCl2 was added to 10 μl sonicated lysate to neutralize the EDTA in the SDS lysis buffer. Tagmentation and amplification was done as described above.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads were then trimmed with Trimmomatic (v0.36) using long TruSeq3-SE.v2 adapters and as essential settings: ILLUMINACLIP:0:30:10, LEADING:3, TRAILING:3, MINLEN:35.
Alignment was done using to strain-specific genomes, generated with g2gtools (v0.2.9) and MGPv5 SNPs and InDels, using STAR (v2.5.4b) with ‘--alignIntronMax 1 --alignEndsType "EndToEnd"’.
Reads were filtered for true pairs and MAPQ>20 with Samtools (v1.9) and duplicates were removed using Picard (v2.8.13) MarkDuplicates.
Peaks were called with MACS2 (v2.2.7.1) using ‘-g mm -B --call-summits --keep-dup auto’. For H3K27ac ‘-q 0.05’, TCF7L2 ‘-q 0.01’ and CTCF ‘-q 0.001’ were used with MACS2.
Genome_build: mm10
Supplementary_files_format_and_content: Sample-wise peaks: For TCF7L2 and H3K27ac summits were widened to peaks with Bedtools (v2.27.1) using ‘slop -b 75’. Peaks were shifted to mm10 coordinates using g2gtools. ENCODE blacklisted regions were removed from the regions.
Supplementary_files_format_and_content: TCF7L2 raw counts: Consensus peak set was formed from the peak centers using modified kernel density estimation -based approach (Tuoresmäki et al. PLoS One 2014;9(4):e96105). csaw was used for counting by using ‘correlateReads’ to determine average fragments length and extending reads to this length before counting.
Supplementary_files_format_and_content: H3K27ac ChIP-R regions & raw counts: ChIP-R (v1.1.0) was used to get group-wise H3K27ac regions (CHIPR-bed files) which were merged using Bedtools to create consensus peak set. csaw (v1.28.0) function ‘correlateReads’ was used to determine average fragments length and reads were extended to this length before counting with csaw.
Supplementary_files_format_and_content: BigWigs: BAM files shifted to mm10 coordinates were downsampled after and merged by group using Samtools. BigWigs were generated using Deeptools (v3.4.2) bamCoverage with '--normalizeUsing CPM --outFileFormat bigwig --binSize 10'.
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| Submission date |
Feb 17, 2022 |
| Last update date |
Dec 11, 2023 |
| Contact name |
Juho Mikael Mononen |
| E-mail(s) |
juho.mononen@uef.fi
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| Organization name |
University of Eastern Finland
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| Department |
Faculty of Health Sciences
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| Lab |
Heikkinen lab
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| Street address |
Yliopistonranta 1E
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| City |
Kuopio |
| ZIP/Postal code |
70210 |
| Country |
Finland |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE196940 |
Genetic determinants of TCF7L2 binding and chromatin availability in mouse liver [ChIP-seq] |
| GSE196942 |
Genetic determinants of TCF7L2 binding and chromatin availability in mouse liver |
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| Relations |
| BioSample |
SAMN26020212 |
| SRA |
SRX14259600 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5905319_H3K27ac_F_B6_1_1.bed.gz |
1.8 Mb |
(ftp)(http) |
BED |
| GSM5905319_counts_H3K27ac_F_B6_1_1.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
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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