 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 12, 2022 |
| Title |
NPCs D868D ChIP SET #2 |
| Sample type |
SRA |
| |
|
| Source name |
NPCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: NPCs
genotype: SETBP1 D868D
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
To perform ChIP experiment 200000 cells for each experimental replicate were used. Cells were cross-linked in 1% formaldehyde for 10’ minutes at room temperature. Cells were lysed in ChIP-lysis Buffer (10mM Tris-HCl 8.0,1% SDS, 5 mM EDTA). Sonication was performed using a Diagenode Bioruptor Pico sonicator (65 cycles, 30” ON-30” OFF, each cycle). Input were collected before precipitation. Samples (two technical replicates for each ChIP) were then incubated overnight at 4° using primary antibodies for H3K27ac, H3K27me3, H3K9me3 and SET in iC buffer from iDeal ChIP‐seq kit for Histones (Diagenode, cod. C01010051). The next day, incubation with magnetic beads was performed on a rotator wheel for 3 hours at 4°. Beads were subsequently washed using iW1, iW2,iW3 and IW4 buffer (Diagenode). ChIP-DNA was then eluted, decrosslinked for 30’ at 37° and 4 hours at 65° and purified using. After quantification 5 ng of DNA were used to build libraries using NebNext-UltraII kit (NEB, cod.). Samples were sequenced paired-end NovaSeq6000 by GeneWiz.
TruSeq
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
NPCD868D_Chip_SET_2
dual index library
|
| Data processing |
FASTQ were first quality checked to evaluate sequence output with using FastQC (Andrews, S. FastQC A Quality Control tool for High Throughput Sequence Data). Reads were then trimmed using Trimmomatic 5. Reads were then aligned to reference genome hg38 using Bowtie2 6 using the --very-sensitive option. Non canonical and M chromosomes were removed from Bam files using Samtools 7 and Picard tools (“Picard Toolkit.” 2019. Broad Institute, GitHub Repository. https://broadinstitute.github.io/picard/) was used to remove PCR optical duplicates, before proceeding with further analysis. Normalized BigWig files for genomics track visualization and for subsequent analysis were generated using deepTools (v3.5.1) 8 bamCoverage with the following parameters --normalizeUsing RPKM --binSize 10 --smoothLength 300 –effectiveGenomeSize --ignoreDuplicates --skipNAs –exactScaling. To obtain a single merged tracks from single experimental replicates for each experimental condition UCSC bigWigMerge was used. Peak calling was performed using MACS2 9 from preprocessed Bam files for each experimental replicate from each condition. For ChIP-seq narrow peaks (H3K27ac), the following parameters were used -f BAMPE --nomodel --qvalue 0.01 --keep-dup all --call-summits using Input for each condition as control sample.
Genome_build: hg38
Supplementary files format and content: macs broadpeak
|
| |
|
| Submission date |
Aug 29, 2022 |
| Last update date |
Sep 12, 2022 |
| Contact name |
Luca Massimino |
| E-mail(s) |
admin@lucamassimino.com
|
| Phone |
+393389039500
|
| Organization name |
Ospedale San Raffaele
|
| Department |
Gastroenterology
|
| Street address |
Via Olgettina, 58
|
| City |
Milano |
| State/province |
MI |
| ZIP/Postal code |
20100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE212252 |
Balanced SET levels favor the correct enhancer repertoire during cell fate acquisition |
|
| Relations |
| BioSample |
SAMN30566522 |
| SRA |
SRX17313996 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6519843_NPCD868D_Chip_SET_2_peaks.broadPeak.gz |
9.4 Kb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
