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| Status |
Public on Nov 09, 2023 |
| Title |
60nM FOXA1, Shifted Band, Rep 2 |
| Sample type |
SRA |
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| Source name |
FOXA1-bound (shifted) synthetic oligonucleotide library at 60 nM FOXA1
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| Organism |
synthetic construct |
| Characteristics |
cell type: not applicable
treatment: 60 nM FOXA1
emsa band: Shifted Band
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| Extracted molecule |
other |
| Extraction protocol |
Synthetic oligonucleotide library was designed to include 3,203 different sequences, each 229bp in length with 193bp variable regions and 18bp primer-binding regions on the two sides. The synthetic oligo library was ordered from Agilent (Product #G7220A).
To amplify the synthetic oligonucleotide library sequences, 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 sample using a NextSeq 2000 (Illumina) instrument.
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| Library strategy |
OTHER |
| Library source |
other |
| 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 EMSA-seq 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 (column 1), nucleotide sequence (column 2), and number of aligned reads or "counts" (column 3) in tabular format.
Library strategy: EMSA-seq
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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 |
GPL32628 |
| Series (2) |
| GSE247431 |
Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1 [EMSA-seq] |
| 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 |
SAMN38190508 |
| SRA |
SRX22470854 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7889881_rawCounts_60nM_Shifted_Rep2.tabular.txt.gz |
401.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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