|
| Status |
Public on Oct 27, 2020 |
| Title |
dex treated callus LFY ChIP-seq Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
callus on CIM for 5 days with dex treatment
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: callus
cultivar: L.er
transgenics: 35S:LFY-GR
mutant: WT
antibody: anti-LFY antibody
|
| Treatment protocol |
5uM of Dexamathasone or mock treatment on the last hour of 5 day CIM incubation was performed
|
| Growth protocol |
Plants were grown on 1/2MS plates for 3 weeks. Roots were harvested from 3-week-old seedlings and put on CIM for up to 5 days with either 1hr of mock or dex treatment
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assay was performed according to Yamaguchi et al., 2014. N. Yamaguchi et al., PROTOCOLS: Chromatin Immunoprecipitation from Arabidopsis Tissues. Arabidopsis Book 12, e0170 (2014).
DNA libraries were generated using the ThruPLEX DNA-seq kit (RUBICON GENOMICS, cat. R400406) and quantified by the NEBNext Library Quant Kit (cat. E7630L). Equal amounts of DNA were pooled from each library with different dual indexing primers and sequenced on a Next seq 500
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Base-calling was performed automatically in Illumina BaseSpace
FASTQ reads were trimmed using TRIMMOMATIC v0.36 (SE -phred33 ILLUMINACLIP:adapters.fasta:2:30:10 LEADING:3 TRAILING:3 MINLEN:50 TOPHRED33)
Sequencing quality metrics were obtained for the raw and trimmed FASTQ files with FASTQC v0.11.5
Trimmed sequencing reads were mapped to Release 10 of the Arabidopsis Genome (TAIR10) using Bowtie2 v2.3.1 with the `--no-unal` parameter
Using samtools v1.7 with the parameters `-F 1804 -q 30 `, reads with a mapping quality score ≧30 were selected that passed Bowtie2 quality checks, and were not called a PCR or optical duplicate by Bowtie2
Duplicated reads were marked using the MarkDuplicates function in Picard tools 2.9.4 (VALIDATION_STRINGENCY=LENIENT REMOVE_DUPLICATES=false ASSUME_SORTED=true)
Regions of the Arabidopsis genome which that have high signal/read counts independent of experiment (i.e noise defined as signal present in the input) were identified using MACS2 (2.1.1.20160309) callpeak with parameters `--keep-dup auto --nomodel --extsize138 --gsize 101274395` on our input samples using q-value ≤10-65. These regions and regions between two such peaks that were ≤2kb apart,were removed from the mapping files using samtools.
Read coverage of each biological replicate was calculated and normalized to 10 million reads mapped per million reads sequenced (10RPM) and coverage was averaged using bedtools.
Peak calling was performed on the ChIP samples using MACS2 callpeak parameters `--keep-dup auto --nomodel --extsize 138 --gsize 101274395` and the input samples controls , using a a summit q-value ≤ 10^-10.
Genome_build: TAIR10
Supplementary_files_format_and_content: sample-specific *bigwig: bigwig normalized to 10RPKM for single replicates
*pooled.bigwig: Bigwig showing 10RPM-normalized coverage after masking of signicant input regions, mean of three replicates
*narrowPeak: BED file in narrowPeak format showing peaks with summit MACS2 Q<1e-10 for Dex treated vs. Mock treated
|
| |
|
| Submission date |
Dec 09, 2019 |
| Last update date |
Oct 27, 2020 |
| Contact name |
Doris Wagner |
| E-mail(s) |
wagnerdo@sas.upenn.edu
|
| Phone |
2158980483
|
| Organization name |
University of Pennsylvania
|
| Department |
Department of Biology
|
| Lab |
Wagner Lab
|
| Street address |
103G Carolyn Lynch Laboratory
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL19580 |
| Series (2) |
| GSE141704 |
LFY is pioneer transcription factor [ChIP-seq] |
| GSE141706 |
LFY is pioneer transcription factor |
|
| Relations |
| BioSample |
SAMN13516312 |
| SRA |
SRX7293446 |
