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| Status |
Public on Jun 08, 2020 |
| Title |
Chuong-ChIP-11 |
| Sample type |
SRA |
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| Source name |
E9 dorsal back feather skin
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| Organism |
Gallus gallus |
| Characteristics |
chip antibody: anti-H3K27ac
skin region: dorsal back
age: E9
skin/appendage layer: whole skin
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| Treatment protocol |
No treatment
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| Growth protocol |
White Leghorn chicken eggs were used. Eggs were incubated in a humidified egg incubator with an automatic egg turner.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Please find detailed protocols for ChIP and Capture-C DNA preparaion in Methods of this publication. RNA extraction for RNA-seq: Total RNAs of indicated tissues were extracted followed by the standard Trizol extraction protocol (Invitrogen, Cat.15596026). ChIPed-DNA preparation for ChIP-seq: The protocol was adapted from Dr. Ruchi Bajpai’s laboratory (Bajpai et al., 2010). 3C library preparation for NG Capture-C: The protocol was adapted from Dr. Wange Lu’s laboratory (Davies et al., 2016; Davies et al., 2017).
For RNA-seq library preparation: TruSeq RNA Prep Kit v2 (Illumina, RS-122-2001/-2002). For ChIP-seq library preparation: NEBNext Ultra End Repair/dA-Tailing Module (New England Biolabs, Cat. E7442S), NEBNext Ultra Ligation Module (New England Biolabs, Cat. E7445S), and KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Cat. KK2601). For NG Capture-C library preparation: 5 µg of sheared 3C-DpnII DNA was used in adaptor ligation for pre-capture library using NEBNext DNA Library Prep Master Mix Set for Illumina (NEB, Cat. E6040). Adaptors were from NEBNext Multiplex Oligos for Illumina (NEB, Cat. E7500). The indexed 3C DNA was amplified using a primer pair of P5 (5’-aatgatacggcgaccaccga) and P7 (5’-caagcagaagacggcatacgagat) and purified using 1.8X AMPure XP beads. To perform the first capture, SeqCap EZ Hybridization and Wash Kit (Roche, Cat. 05634261001) was used. For one library, the indexed sequencing library, 5 µg of a Cot-1 DNA equivalent chicken Hybloc DNA (Applied Genetics Lab, Cat. CHB), 1 µl of 1xGen Universal Blocking Oligo—TS-p5 (1 nmole/µl), and 1 µl of 1xGen Universal Blocking Oligo—TS-p7(6nt) (1 nmole/µl) were mixed well and dried completely in a low retention PCR tube (Axygen, Cat. PCR-02-L-C). The dried pellet was then resuspended in Nimblegen Hybridization Buffer and Component A, mixed with 2 µl of a capture probe pool (3 pmole in total), and incubated for hybridization at 47℃ for 72 hours. After the first hybridization, biotinylated-probe-hybridized DNA (captured DNA) was mixed with 40 µl of prepared streptavidin magnetic beads (Invitrogen, Cat. 65001) and incubated at 47℃ for 45 min. The mixture was further washed using the SeqCap Wash Kit. The captured DNA was then purified using 1.8X AMPure XP beads and eluted in final 44 µl of nuclease-free water and amplified using the P5/P7 primer pair for 12 cycles. This 1st-captured DNA was finally cleaned up with 1.8X AMPure XP beads and eluted in 25 µl of water. To perform the second capture, the 1st-captured DNA was mixed with components described in the first capture part and incubated for hybridization at 47℃ for 24 hours. The double-captured DNA was then washed, pulled down by using the streptavidin beads, purified, amplified, and cleaned up as described before.
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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 |
RNA-seq analysis. The RNA-seq analysis pipeline was adapted according to the publication (Trapnell et al., 2012). Manipulation. Tools: fastq_groomer (Galaxy Version 1.1.1) and fastq_trimmer_by_quality (window size 3; min score >= 20). Mapping. Tool: TopHat2 (Galaxy Version 2.1.1). Quantification and differentially expressed gene analysis. Tools: Cufflinks package (Trapnell et al., 2010)–including Cufflinks (Galaxy Version 2.2.1.2), Cuffmerge (Galaxy Version 2.2.1.1), Cuffquant (Galaxy Version 2.2.1.1), and Cuffdiff (Galaxy Version 2.2.1.3). Deeptools bamCoverage (Galaxy Version 3.1.2.0.0) was used to normalize the reads to 1X sequencing depth and wig_to_bigwig (Galaxy Version 1.1.1) was used to generate bigWig files of read coverages.
ChIP-seq analysis. Manipulation. Tools: fastq_groomer (Galaxy Version 1.1.1), fastq_quality_filter (Galaxy Version 1.0.2), fastq_trimmer_by_quality (Galaxy Version 1.1.1; window size 3; min score >= 20). Mapping. Tool: bowtie2 (Galaxy Version 2.3.4.2; genome: UCSC galGal4, --very-sensitive). Filtering. Tools: samtool_filter2 (Galaxy Version 1.8; -q 1), samtools_sort (Galaxy Version 2.0.1), samtools_rmdup (Galaxy Version 1.0.1; BAM is single-end). Peak-calling. Tools: MACS2 (Galaxy Version 2.1.1.20160309.4; for H3K27ac-ChIP, --gsize 1065365425 --nomodel --extsize 147 --bdg --broad --broad-cutoff 0.1; for CTCF/KLF-ChIP, --gsize 1065365425 --nomodel --extsize value obtained from macs2_predictd tool) and macs2_bdgcmp (Galaxy Version 2.1.1.20160309.0; -m FE and -m logLR -p 0.00001). To normalize MACS2 results from different libraries for comparison, we followed a “Build Signal Track” instruction from https://github.com/taoliu/MACS/wiki/Build-Signal-Track#Run_MACS2_bdgcmp_to_generate_foldenrichment_and_logLR_track. Convert format. Tool: wig_to_bigWig (Galaxy Version 1.1.1).
NG Capture-C analysis. Double Capture-C results were analyzed according to the publication (Davies et al., 2016). The analysis pipeline was well documented and can be downloaded from “https://github.com/Hughes-Genome-Group/captureC/releases/download/VS2.0/UserManualforCaptureCanalysis.pdf". In brief, 1) download the CaptureC analyser package including CCanalyser3.pl, dpngenome3_1.pl, dpnII2E.pl from “https://github.com/Hughes-Genome-Group/captureC/releases", 2) generate in silico DpnII digested chicken genome ver. Ensemble galGal4 Release 72 with alpha and beta keratins, 3) perform adapter trimmer using the tool trim_galore (Dr. Felix Krueger at the Babraham Institute), 4) Merge overlapping reads by using the tool FLASH (Magoc and Salzberg, 2011), 5) in silico DpnII digest the FLASH-merged reads; 6) install Bowtie 1.2 (Langmead et al., 2009), 7) build up bowtie index using the original genome used in the Step 2, 8) align digested FLASH-merged reads (output of Step 5) using Bowtie1.2, and 9) run CCanalyser3.pl. wig_to_bigwig (Galaxy Version 1.1.1) was used to generate bigWig files from wig files of CCanalyser3 outputs.
Genome_build: UCSC Genome Browser assembly ID: galGal4
Supplementary_files_format_and_content: Bigwig files were generated using the wig/bedgraph_to_bigwig tool. Detail processes of RNA-seq, ChIP-seq, and NG Capture-C are described above.
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| Submission date |
Aug 22, 2019 |
| Last update date |
Jun 09, 2020 |
| Contact name |
Ya-Chen Liang |
| E-mail(s) |
ya-chen.liang@med.usc.edu
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| Phone |
13234422985
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| Organization name |
University of Southern California
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| Department |
Department of Pathology
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| Lab |
Dr. Cheng-Ming Chuong
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| Street address |
2011 Zonal Ave., HMR 304A
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL19787 |
| Series (1) |
| GSE136224 |
Folding Keratin Gene Clusters During Skin Specification |
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| Relations |
| BioSample |
SAMN12623047 |
| SRA |
SRX6752378 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4042864_ChIP-H3K27ac_E9_Fe_normalized-FE_ChIP11-15.bigwig |
449.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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