|
| Status |
Public on Aug 29, 2018 |
| Title |
rluc hmg3 rep3 |
| Sample type |
SRA |
| |
|
| Source name |
whole worms
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain/background: N2
developmental stage: L4
tissue: whole body
sirna/dsrna: HT115 (D3) bacteria containing L4440 vector with DNA targeting Renilla luciferase
|
| Treatment protocol |
For F1 RNAi experiments, worms were synchronized by bleaching and L4 larvae were put on RNAi plates and grown at 15ºC until most of the progeny animals reached L4 stage.
|
| Growth protocol |
C. elegans strains were maintained on OP50 bacteria and NGM agar plates at 15ºC. Worms were transferred to fresh food once a week.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Synchronized L4 animals were washed 5 times in M9 buffer and collected on ice. Nuclei were isolated using a glass Dounce homogenizer with 50 strokes tight-fitting insert in buffer A (15 mM Tris–HCl pH7.5, 2 mM MgCl2, 340 mM sucrose, 0.2 mM spermine, 0.5 mM spermidine, 0.5 mM phenylmethanesulfonate [PMSF], 1mM DTT, 0.1% Triton X-100 and 0.25% NP-40 substitute). The debris were removed by spinning at 100×g for 5 min and nuclei were counted by Methylene blue staining. 100.000 nuclei per sample were pelleted by spinning at 1000×g for 10 min and proceeded immediately to transposition step of the ATACseq protocol.
Library preparation was done following a protocol published in Buenrostro et al. 2013. Cell pellet was resuspended in the transposase reaction mix (25 μL 2× TD buffer, 2.5 μL transposase) (Illumina Nextera DNA Library preparation kit, #FC-121-1030). The transposition reaction was carried out for 60 min at 37ºC. The samples were purified using Zymo DNA Clean & Concentrator kit; following purification, library fragments were amplified using NEBNext PCR master mix and custom Nextera PCR primers. Libraries were amplified for a total of 12 to 14 cycles and sequenced using paired-end-sequencing length of 75 nucleotides using NextSeq 500/550 High Output v2 kit (Illumina) on a NextSeq500 machine (Illumina) following manufacturer's protocol.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Processed data file: hotFiltered_rlucWholeCat_steric38.bed.bigWig
Processed data file: hmg3_totalCounts.txt
|
| Data processing |
ATAC-seq reads were trimmed for adapters using flexbar v2.5 (-f i1.8 -u 10 -ae RIGHT -at 1.0) and mapped with bowtie2 v2.0.2 in default paired-end mode and restricting pair distances to 1500 (-X 1500 -- no-discordant) to version hg19 of the human genome or ce10 of the worm genome followed by removal of multimappers. PCR duplicates were removed using Picard Tools MarkDuplicates v1.90 and reads were converted to .bed format using bedtools bamToBed v2.23. Pairs were filtered out if they mapped to the same strand, to different chromosomes, or if the 5’end coordinate of the – strand read was less than or equal to the 5’end coordinate of the + strand read. Mapped pairs were split into single reads and converted to a 38-bp fragment reflecting the theoretical minimal spacing required for a transposition event by Tn5 transposome using bedtools slop on the read 5’ends (-l 15 -r 22). Replicates were concatenated after confirming high concordance. C. elegans datasets were further filtered for reads from the rDNA loci as well as those mapping to regions corresponding to transgenic reporter constructs existing in the strains and corresponding to sequences used in the RNAi vectors.
Peaks were called on concatenated processed bed files using JAMM peakcaller v1.0.7.5 (-f38 –b 100 –e 1.75). The resulting “all” (for worm) or “filtered” (for human) peaks output by JAMM for each condition (control, ssrp1 knockdown, and supt16h knockdown for human, or rluc, hmg3, and hmg4 for worms) were concatenated and then merged with bedtools merge v2.23. The merged "all" peaks for worm were then further filtered for a minimum width of 25 bp.
Merged peaks were then counted for the number of processed reads that intersected them from each replicate of each experimental condition using bedtools coverage v2.23 (-counts). Count tables were then normalized (method = “TMM”) and subjected to differential analysis (exactTest) using edgeR. Peaks were called as differential with a q-value cutoff of 0.01 (adjust=”BH”).
bedGraph files were generated from replicate concatenated processed ATAC-seq reads .bed files using bedtools genomecov v2.23 (-scale 1/(library size - chrM reads/100000)) which were then converted to .bigWig files using the ENCODE program bedGraphToBigWig.
RNA-seq reads were trimmed for adapters and mapped with STAR.
RNA-seq count tables were generated using quasR and differential expression determined using DESeq2.
Genome_build: ce10
Supplementary_files_format_and_content: *tsv: Tab-delimited text files; RNA-seq count tables.
Supplementary_files_format_and_content: *bigWig: Library-normalized bigWig files of processed ATAC-seq reads.
Supplementary_files_format_and_content: *txt: Tab-delimited text files; Count tables for merged peaks across all replicates separately.
|
| |
|
| Submission date |
Jul 23, 2017 |
| Last update date |
Aug 29, 2018 |
| Contact name |
Scott Allen Lacadie |
| E-mail(s) |
[email protected]
|
| Organization name |
Max Delbrück Center for Molecular Medicine
|
| Department |
Berlin Institute for Medical Systems Biology
|
| Lab |
Ohler
|
| Street address |
Robert-Rössle-Str. 10
|
| City |
Berlin-Buch |
| ZIP/Postal code |
13092 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19757 |
| Series (1) |
| GSE98758 |
Genome-wide DNA accessibility maps and differential gene expression using ChIP-seq, ATAC-seq and RNA-seq for the human secondary fibroblast cell line hiF-T and whole worms with and without knockdown of FACT complex |
|
| Relations |
| BioSample |
SAMN07407125 |
| SRA |
SRX3029123 |
