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| Status |
Public on Nov 22, 2023 |
| Title |
dmso_atac_explant_rep2 |
| Sample type |
SRA |
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| Source name |
Primary Neural Crest Cells
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| Organism |
Gallus gallus |
| Characteristics |
cell type: Primary Neural Crest Cells
Stage: HH9 stage
treatment: Neural crest cells explants cultured on fibronectin-coated tissue culture plates treated with vehicle (DMSO)
transposase: Tn5 enzyme (Illumina 20034210)
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| Treatment protocol |
NFs were dissected from six HH9 embryos and cultured as described above in paired fashion in media containing DMSO or 40 uM (R)-GNE-140 (Selleck Chemicals, S6675) (n=6 neural folds per control/treatment). This experiment was performed with two biological replicates. Explants were cultured for 12 hours using previously described settings, at which point they were collected from the plate by incubation with Accumax (Innovative Cell Technologies, AM105) for 10 minutes at 37°C. The single cells from each condition/sample were subjected to the ATAC-seq protocol (described above).
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| Growth protocol |
Prior to embryo dissection, tissue cultureware were coated with 25 ug/ml fibronectin (Sigma, F1141) and incubated for 45 min in a 37°C humidified incubator maintained at 5%CO2 atmosphere. After incubation, the fibronectin solution was removed from the cultureware and DMEM (Sigma, D6046) supplemented with 10% FBS (Thermo Fisher Scientific, 10-437-028) was added. NCC explants were obtained by thinly micro-dissecting dorsal neural folds from HH9 (6-7 somite) embryos in Ringer’s solution as previously described (http://www.genome.org/cgi/doi/10.1101/gr.249680.119). Each dissected neural fold was then aspirated in a minimal amount of Ringer’s solution (2.5 ul) and placed in a well of the fibronectin-coated tissue cultureware containing media. The explants were incubated for at least 12 hours under normoxic conditions in a humidified 37°C incubator maintained at 5% CO2 atmosphere. The duration of incubation was determined by the appropriate conditions pertaining to specific experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
NFs were dissected from HH9 embryos as described in the CUT&RUN protocol (n=2 embryos, n=4 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.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ATAC-seq
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| 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.
The R package DiffBind (version 2.14.0) was used to perform the differential accessibility analysis between DMSO and (R)-GNE-140 ATAC-seq samples. The consensus control peakset between DMSO replicates (generated using BEDTools as described in the “ATAC-seq Data Analysis” section) was first filtered to include only peaks that map to chromosomes (not contigs). This consensus peakset was then used to count reads from each sample/replicate. Binding affinity matrix scores were reported after performing a TMM normalization using full library size. The consensus peakset was filtered to remove peaks with low read counts (filter=50 with filterFun applied to the scores of each interval). The comparison between samples was set up to account for the fact that the DMSO and (R)-GNE-140 samples from each set of biological replicates were paired (i.e., tissue came from the same set of embryos for each condition). This was done by specifying the block=DBA_REPLICATE argument when setting up contrasts using the dba.contrast() function. Differential accessibility statistical analysis was performed using the DBA_EDGER method. Assignment of differential peaks to their closest gene was done using the R package ChIPseeker.
Assembly: Ensembl galGal6
Supplementary files format and content: bw file
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| Submission date |
Mar 27, 2023 |
| Last update date |
Nov 22, 2023 |
| Contact name |
Marcos Simoes-Costa |
| Organization name |
Boston Children's Hospital
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| Department |
Pathology
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| Street address |
300 Longwood Ave
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19787 |
| Series (2) |
| GSE228342 |
Histone lactylation couples cellular metabolism with the activation of developmental gene regulatory networks [R-GNE-140 ATAC-seq] |
| GSE228343 |
Histone lactylation couples cellular metabolism with the activation of developmental gene regulatory networks |
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| Relations |
| BioSample |
SAMN33942642 |
| SRA |
SRX19789004 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7119216_dmso_atac_explant_rep2_nodups.bw |
92.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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