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| Status |
Public on Nov 09, 2023 |
| Title |
A549, FOXA1, ChIP-ISO ChIP, biol rep 1 |
| Sample type |
SRA |
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| Source name |
A549
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| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
cell type: Human lung carcinoma
treatment: ISO library integrated at AAVS1
antibody: FOXA1 (GeneTex GTX100308)
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| Treatment protocol |
None
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| Growth protocol |
Ham's F-12K (Kaighn's), 10% fetal bovine serum, 1% penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with formaldehyde (1% final concentration) for 10 minutes at room temperature. Crosslinking was quenched with glycine (0.125M final concentration) for 5 minutes at room temperature. After cell and nuclei lysis, chromatin was fragmented in Diagenode Pico with a circulating water bath at 4°C using the Shear and Go Easy Mode setting for 3 cycles (30 seconds on followed by 30 seconds off).
To amplify the integrated ChIP-ISO library sequences while excluding other genomic DNA, including native CCND1e, we performed two rounds of PCR amplification with a BbsI digestion step in between. The primer pairs for the first round of PCR contain regions annealing to the CCND1e (outside the 193bp library sequence) at the 3’-ends, partial Illumina TruSeq adaptor sequences at the 5’-end and 0-3 random nucleotide spacers in between to increase sequence complexity. Primers with different numbers of spacers are mixed in equimolar ratio for the first round of PCR. Preliminary PCR tests were performed to decide the optimal cycle number that keeps the PCR reactions in exponential phase. We used 23-25 cycles for our first round of PCR. For the first round of PCR, 30 μl of ChIP DNA was amplified in a 100 μl PCR reaction using NEBNext Ultra II Q5 master mix. The PCR products were purified with AMPure XP beads with a beads to DNA ratio of 0.9. 15 μL of the purified PCR product was digested with 20 U of BbsI in a 30 μL reaction at 37°C for two hours. The digestion products were purified with AMPure XP beads with a beads to DNA ratio of 0.9, followed by the second round of PCR. The primer pairs for the second round of PCR contain the rest of the Illumina TruSeq adaptor sequences and sample indexes. For the second round of PCR, 2 μl of purified digestion product was amplified in a 50 μl PCR reaction for 8 cycles using NEBNext Ultra II Q5 master mix. The PCR products were purified with AMPure XP beads with a beads to DNA ratio of 0.8. Quality control was conducted with TapeStation (Agilent). 30 million paired-end 150bp reads were obtained for each ChIP-ISO and input sample using a NextSeq 2000 (Illumina) instrument.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Basecalls were performed using DRAGEN BCL Convert (v3.8.4).
Paired-end reads were filtered using fastp (Version 0.23.2) according to default settings.
First three nucleotides were trimmed from each read using cutadapt to remove the 0-3 random nucleotides introduced by the amplicon primers.
Paired-end reads were aligned to a FASTA file containing all ChIP-ISO library sequences in their forward and reverse orientation using BWA-MEM2
BAM alignments containing at least 2 mismatched nucleotides were filtered out using BAMtools
Number of sequences (counts) aligning to each library sequence were determined using the "count occurences of each record" tool in Galaxy
Assembly: hg38
Supplementary files format and content: Raw counts files including sequence ID and number of aligned reads or "counts" in tabular format.
Library strategy: ChIP-ISO
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| Submission date |
Nov 09, 2023 |
| Last update date |
Nov 09, 2023 |
| Contact name |
Lu Bai |
| E-mail(s) |
[email protected]
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| Organization name |
Penn State University
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| Street address |
406 South Frear Building
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE247411 |
Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1 [ChIP-ISO] |
| GSE247432 |
Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1 |
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| Relations |
| BioSample |
SAMN38194668 |
| SRA |
SRX22478371 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7889156_A549_ChIP_ISO_lib1p2_FOXA1_rep1_rawCounts.tabular.txt.gz |
29.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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