 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 04, 2019 |
| Title |
Gli2_ChIPSeq Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
E17.5 intestine
|
| Organism |
Mus musculus |
| Characteristics |
age: E17.5
tissue: intestine
genotype/variation: Sufu;Spop double knockout
chip antibody: Gli2 (homemade)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
E17.5 intestines were dissected, cut into small pieces, and crosslinked in solution A (1% formaldehyde, 50mM Hepes-KOH, 100 mM NaCl, 1mM EDTA, 0.5 mM EGTA) for 20 minutes at room temperature. 1/20 volume of 2.5 M glycine was added to quench formaldehyde. Tissues were rinsed in ice-cold PBS and dounced in Wheaton tissue grinder. Cell suspensions were passed through a 100μm cell strainer to remove connective tissues, then centrifuged at 4 °C at 2500 × rcf for 3 min, followed by a wash in PBS. Gli2, H27K27ac, or H3K36me3 antibodies were incubated with pre-blocked magnetic beads in 250μL total volume for 8 hours at 4 °C, then washed and resuspended in 100μL 0.5% BSA (w/v) solution. Cell pellets were lysed by sequentially resuspending and centrifuging in LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100), and LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA). Pellet was resuspended in LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine) and sonicated into 200-400bp fragments. Sonicated lysates were diluted with LB3. After saving 50μL for input control, they were split into tubes containing 10% v/v Triton X-100 with Gli2, H3K27ac, or H3K36me3 antibody, and incubated overnight. Beads were washed 6 times in RIPA buffer (50 mM Hepes-KOH, pH 7.5; 500 mM LiCl; 1mM EDTA; 1% NP-40 or Igepal CA-630; 0.7% Na-Deoxycholate), and once in TBS (20 mM Tris-HCl, pH 7.6; 150 mM NaCl). Protein-DNA complexes were eluted and crosslink reversed with 200 μL of elution buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA; 1% SDS). The 50μL input lysate was also included in this step. Eluted DNA was diluted with 200 μL of TE, and treated with RNaseA for 30 min at 37°C, and subsequently with proteinase K for 2 hours at 55°C. Phenol:chloroform:isoamyl alcohol was used to extract DNA.
Libraries were prepared according to manfacturer's instructions for the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Gli2_int_merged_nodup.BED
|
| Data processing |
Adaptor sequences were trimmed using Trimmomatic 0.36 with adaptor sequence file TruSeq3-PE-2.fa
Reads were aligned to the mm9 reference genome using default parameters of BWA MEM
Aligned reads were filtered using bamtools to exclude unmappped reads, mapping quality < 5, and not proper pairs.
ENCODE blacklist regions in mm9 were filtered out using bedtools
Peaks were called using MACS2 with q = 0.05. --broad option was used for histone marks
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files were generated using bamCoverage tool from DeepTools
Supplementary_files_format_and_content: Peaks from individual replicates were merged using "cat" shell command. "mergeBed" tool from BedTools was used to remove duplicate peaks and generate the final BED file specifiying the genomic position of the peaks.
|
| |
|
| Submission date |
Sep 11, 2017 |
| Last update date |
Jun 04, 2019 |
| Contact name |
Tae-Hee Kim |
| Organization name |
The Hospital for Sick Children
|
| Department |
Developmental & Stem Cell Biology
|
| Street address |
686 Bay Street. Room 16.9709
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 0A4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE103690 |
GLI2-mediated mesenchymal Hh signaling induces stem cell niche signals in intestinal development and tumorigenesis [ChIPSeq] |
| GSE103691 |
GLI2-mediated mesenchymal Hh signaling induces stem cell niche signals in intestinal development and tumorigenesis |
|
| Relations |
| BioSample |
SAMN07629369 |
| SRA |
SRX3176061 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2779301_7A_gli2_int1_filtered_intersect.bigwig |
84.8 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 |
|
|
|
|
 |
