 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 22, 2023 |
| Title |
atac_nf_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Primary Neural Crest Cells
|
| Organism |
Gallus gallus |
| Characteristics |
cell type: Primary Neural Crest Cells
Stage: HH9 stage
genotype: wild-type
treatment: embryos developed in ovo until HH9
transposase: Tn5 enzyme (Illumina 20034210)
|
| Treatment protocol |
The collected embryos were wild-type.
|
| Growth protocol |
Fertilized White Leghorn eggs were purchased from the University of Connecticut (Department of Animal Sciences). The eggs were incubated at 37°C until the embryos reached HH9 (6-7 somites) stage of development.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
NFs were dissected from HH9 embryos as described in the CUT&RUN protocol (n=4 embryos, n=8 NFs) and dissociated in Accumax (Innovative Cell Technologies, AM105) for 30 min at room temperature to achieve a single cell suspension.
ATAC-seq was performed as previously described (https://doi.org/10.1038/nmeth.4396). Briefly, dissociated cells were collected by centrifugation at 500 rcf for 5 min in a swinging bucket centrifuge and washed in resuspension buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2. After washing, the cells were resuspended in lysis buffer composed of the resuspension buffer with 0.1% NP-40 (Sigma, I8896), 0.1% Tween-20 (Sigma, P1379), and 0.01% Digitonin (Millipore, 300410). The samples were incubated on ice for 3 min, at which point the lysis buffer was washed out with resuspension buffer containing only 0.1% Tween-20. Upon centrifugation at 500 rcf for 5 min, the supernatant was aspirated, and nuclei were resuspended in 50 ul of transposition mixture containing 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 10% Dimethyl Formamide (Sigma, D4551), 1X PBS, 0.1% Tween-20, 0.01% Digitonin, and 5 ul Tn5 transposase (Illumina, 20034210). Transposition was performed for 1 hour at 37°C in a thermal mixer. The pre-amplification DNA was purified using the Qiagen Minelute Reaction Cleanup Kit (Qiagen, 28204). Library amplification PCR was performed using the Q5® High-Fidelity 2X Master Mix (NEB, M0492S). Library size selection and cleanup was carried out using Ampure XP beads (Beckman Coulter, A63881). Prepared libraries were run on an Agilent 4200 Tapestation with D5000 Reagents and D500 ScreenTape for quality control and final libraires were pooled to equimolar concentrations using the NEB Library Quantification Kit.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ATAC-seq
|
| Data processing |
For ATAC-seq data analysis, paired-end sequencing reads were trimmed using Cutadapt (v2.10) using the following arguments “cutadapt -a CTGTCTCTTATACACATCT -A AGATGTGTATAAGAGACAG --minimum-length=25”.
Bowtie2 (v2.4.2) was used to align trimmed reads to the reference chicken galGal6 assembly using the following arguments “--no-unal --no-mixed --no-discordant -X 2000”. Duplicate reads were then marked by the Picard MarkDuplicates tool and SAMtools was used to filter BAM files to discard unmapped reads, reads which were not the primary alignment, reads failing platform/vendor quality checks, and PCR/optical duplicates (-F 1804 -f 2).
MACS2 was used to call peaks genome-wide with a q value cutoff equal to 0.05 with the following arguments “-f BAMPE -g 1218492533 -q 0.05 call-summits --nomodel --shift 37 --extsize 73”. Consensus peaksets between biological replicates were determined by intersecting the peaksets of each replicate by using the BEDTools intersect function and only keeping peaks present in both replicates.
Similar to CUT&RUN data processing, featureCounts was used to generate peak count matrices for specific BAM files (when applicable) using the following arguments “--fracOverlap 0.5 --minOverlap 5 -p” and the edgeR package was used to first obtain TMM-normalized effective library sizes that were then used to normalize raw peak counts with the cpm() function. Peaks were annotated based on their closest gene using the R package ChIPSeeker and tornado and profile plots were generated using the DeepTools package. The bamCoverage function of DeepTools was used to generate RPGC-normalized BigWig files, for visualization of normalized read pile-up in a genome browser (IGV v2.13.0), using the following arguments “--outFileFormat bigwig --binSize 5 --numberOfProcessors 10 --normalizeUsing RPGC --effectiveGenomeSize 1218492533 --extendReads”.
Assembly: Ensembl galGal6
Supplementary files format and content: bw file
|
| |
|
| Submission date |
Mar 27, 2023 |
| Last update date |
Nov 22, 2023 |
| Contact name |
Marcos Simoes-Costa |
| Organization name |
Boston Children's Hospital
|
| Department |
Pathology
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL19787 |
| Series (2) |
| GSE228340 |
Histone lactylation couples cellular metabolism with the activation of developmental gene regulatory networks [NCC ATAC-seq] |
| GSE228343 |
Histone lactylation couples cellular metabolism with the activation of developmental gene regulatory networks |
|
| Relations |
| BioSample |
SAMN33942532 |
| SRA |
SRX19789002 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7119210_atac_nf_rep2_nodups.bw |
77.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
