|
| Status |
Public on Aug 29, 2018 |
| Title |
gAtac_rluc1: gonad ATAC-seq rep1 |
| Sample type |
SRA |
| |
|
| Source name |
worm gonads
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
genotype/variation: N2
tissue: worm gonads
passages/stage: L4
|
| 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. 20 gonads per sample were isolated by hand. 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% Trition X-100 and 0.25% NP-40 substitute). The debris were removed by spinning at 100×g for 5 min and the nuclei 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 HiSeq 4000 |
| |
|
| Description |
ATACseq
|
| 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 will as those mapping to regions corresponding to transgenic reporter constructs existing in the strains and corresponding to sequences used in the RNAi vectors.
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.
UMIs from RNA-seq reads were extracted with UNI-tools followed by read trimming with fastx_trimmer and mapping with STAR. Mapped files were deduplicated with UNI-tools and remapped with STAR to generate bam and bedGraph files
RNA-seq normalized bedGraph files were converted to bigWig format with bedGraphToBigWig and replicates were averaged using bamCompare from the deeptools suite.
RNA-seq count tables were generated using RSEM and differential expression determined using DESeq2
UMIs from ChIP-seq reads were extracted using UMI-tools followed by read trimming with fastx_trimmmer and mapping with Bowtie2.
Uniquely mapping ChIP-seq reads were converted to bed files of the full fragment lengths then to library normalized bigWig files using bamCoverage from the deepTools suite. BigWig files were then normalized to input controls using bamCompare from the deepTools suite with --ratio substract for worm and --ratio ratio for human
Windows around annotated transcription start sites were counted for intersections with full-paired-end-fragment ChIP-seq reads using bedtools intersect and windows containing significant signal determined using DESeq2
Genome_build: ce10, hg19
Supplementary_files_format_and_content: library-normalized bigWig files of processed ATAC-seq reads, mapped RNA-seq reads, and input-normalized ChIP-seq reads
Supplementary_files_format_and_content: Gene count tables for RNA-seq reads and promoter-window count tables for full-paired-end-fragment ChIP-seq reads
|
| |
|
| Submission date |
May 03, 2018 |
| Last update date |
Aug 29, 2018 |
| Contact name |
Scott Allen Lacadie |
| E-mail(s) |
scott.lacadie@mdc-berlin.de
|
| 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 |
GPL22765 |
| 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 |
SAMN09041801 |
| SRA |
SRX4029336 |
