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| Status |
Public on Nov 14, 2020 |
| Title |
ZNF416_FLAG_rep2 |
| Sample type |
SRA |
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| Source name |
lung
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| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90 human lung fibroblasts
antibody: ZNF416-FLAG
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| Treatment protocol |
none
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| Growth protocol |
IMR90's were grown in DMEM supplemented with 10% FBS and antibiotics. Lentiviral particles were used to generate stably expressing ZNF416-FLAG IMR90's. After transduction, IMR90s expressing ZNF416-FLAG were selected using puromycin. Surviving cells were then expanded for ChIPseq.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ZNF416-FLAG expressing IMR90 cells were subjected to ChIP-seq for FLAG as previously described. Briefly, IMR90 cells (n=2 biological replicates) stably expressing ZNF416-FLAG were cross-linked with 1% formaldehyde (Sigma Aldrich, St. Louis, MO, USA) for 10 minutes, followed by quenching with 125 mM glycine (Sigma Aldrich, St. Louis, MO, USA) for 5 minutes at room temperature. Fixed cells were washed with 1X TBS (Thermo Fisher Scientific, Waltham, MA, USA). IMR90 cells were then resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 minutes. The lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2) and incubated for 20 minutes at 37 °C in the presence of MNase. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% Sodium deoxycholate), the lysate was sonicated for 10 minutes (30 sec-on / 30 sec-off) in a Bioruptor instrument (Diagenode, Denville, NJ, USA) and then centrifuged at 15,000 rpm for 10 minutes. The cleared supernatant equivalent to about 15 million cells was incubated with 30 µl of prewashed anti-FLAG M2 magnetic beads (Sigma, Catalogue #: M8823) (Sigma Aldrich, St. Louis, MO, USA) on a rocker overnight. The beads were extensively washed with ChIP buffer, high salt buffer, LiCl2 buffer, and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65 °C overnight. DNA was treated with RNase A and Proteinase K (Qiagen, Valencia, CA, USA) and then purified using a Min-Elute PCR purification kit (Qiagen, Valencia, CA, USA). Sequencing libraries were prepared from 5 - 10 ng of ChIP and input DNA with the ThruPLEX® DNA-seq Kit V2 (Rubicon Genomics, Ann Arbor, MI) and were sequenced to 51 base pairs from both ends using an Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Paired-end fastq files were aligned to the hg19 build of the human genome using Bowtie2 using the default settings. SAM files were converted to BAM and were sorted by chromosomal-coordinates using Picard SortSam. Duplicates were removed using Picard MarkDuplicates. BAM files were then used to call peaks using MACS2 using default settings with a q threshold of 0.05. To generate BigWig files, deepTools BamCoverage was used with default settings and a bin-size of 10 base pairs. Integrative Genome Viewer (IGV) was used to visualize BigWig files on the hg19 genome track. Homer was used to annotate ZNF416 binding sites to the nearest TSS as described above. Gene ontology of ZNF416 target genes was completed as described above.
Genome_build: hg19
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| Submission date |
Nov 13, 2020 |
| Last update date |
Nov 15, 2020 |
| Contact name |
Aditya Bhagwate |
| E-mail(s) |
bhagwate.adityavijay@mayo.edu
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| Organization name |
Mayo Clinic
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| Street address |
200 1st Street SW
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| City |
Rochester |
| State/province |
MN |
| ZIP/Postal code |
55905 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE161446 |
ZNF416 is a pivotal transcriptional regulator of fibroblast mechano-activation [ChIP-seq] |
| GSE161448 |
ZNF416 is a pivotal transcriptional regulator of fibroblast mechano-activation |
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| Relations |
| BioSample |
SAMN16793269 |
| SRA |
SRX9505511 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4907356_ZNF416_rep2.bigwig |
621.2 Mb |
(ftp)(http) |
BIGWIG |
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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