|
| Status |
Public on Nov 27, 2017 |
| Title |
Sample_43844 |
| Sample type |
SRA |
| |
|
| Source name |
NB490 cell line
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NB490
chip antibody: None
sample type: Input
|
| Growth protocol |
NB490 cells (kindly provided by Nabeel Bardeesy) were grown in DMEM supplemented with 10% FBS and 50 mg/mL gentamycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from the NB490 cell line using the truChIP Chromatin Shearing Reagent Kit (Covaris;520154). Chromatin was sheared in 1 mL millitubes (Covaris;520135) using a Covaris S200 (5% duty cycle, intensity of 4, 200 cycles per burst, for 8 minutes) to obtain chromatin fragments 100 ? 700 base-pairs in length. 50 ug of fragmented chromatin was incubated overnight with 2.5 uL of rabbit anti-PDX1 antibody (Millipore;07-696). Complexes were isolated using 50 uL of Dynabeads (Invitrogen;10004D) previously blocked with BSA (1 mg/mL).
2 ng of immuno-precipitated DNA was prepared as a standard Illumina library using the ThruPLEX DNA-seq Kit (Rubicon), according to manufacturer?s protocol. Samples were PCR-amplified and pooled. Final libraries were checked for quality and quantity by TapeStation (Agilent) and qPCR using Kapa?s library quantification kit for Illumina Sequencing platforms (Kapa Biosystems;KK4835). They were clustered on the cBot (Illumina) and sequenced 2 samples per lane on a 50-cycle single-end on a HiSeq 2000 in High Output mode using version 3 reagents.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were downloaded and concatenated to yield one .fastq.gz file for each sample, and FastQC v0.10.0 was used to assess data quality for each file.
Reads were aligned to mm10 (UCSC) using bowtie2 with the following parameter settings: bowtie2 --phred33 -p 2 --very-sensitive -x genome -U Sample_43843_R1.fastq -S /ccmb/BioinfCore/Projects/Crawford_howcraw_mceachin_HI_CS3_1446/mapped_reads/Sample_43843_R1_k2.sam
MACS2 (v2.0.10) was used to identify peaks with the following parameter settings: macs2 callpeak -t /ccmb/BioinfCore/Projects/Crawford_howcraw_mceachin_HI_CS3_1446/mapped_reads/ChIPR_A/POOLREP_ChIPR_A.bam -c /ccmb/BioinfCore/Projects/Crawford_howcraw_mceachin_HI_CS3_1446/mapped_reads/INPUT_A/POOLINPUT_A.bam -n POOLREP_MACS_ChIPR_A_q0.01 -f BAM -g mm -B -q 0.01. Blacklisted islands were removed from the list of peaks (https://sites.google.com/site/anshulkundaje/projects/blacklists)
Peaks were annotated by HOMER (v4.6) for their locations relative to nearby genes.
Bigwig files were generated by counting read pile-ups using MACS.
Genome_build: mm10
Supplementary_files_format_and_content: BigWig
|
| |
|
| Submission date |
Dec 08, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Richard C McEachin |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Michigan
|
| Department |
DCM&B
|
| Street address |
2800 Plymouth Road
|
| City |
Ann Arbor |
| State/province |
United States |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE91054 |
PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance [ChIP-seq batch2] |
| GSE91056 |
PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance |
|
| Relations |
| BioSample |
SAMN06124607 |
| SRA |
SRX2405675 |
