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Sample GSM5074265 Query DataSets for GSM5074265
Status Public on Feb 11, 2021
Title 198CS18
Sample type SRA
Source name seedling
Organism Arabidopsis thaliana
Characteristics cultivar: Col-0
genotype: CAMTA1-FLAG in camta123
tissue: seedling
photoperiod: long days (16L/8D)
temperature: 22C
replicate: single
fragmentation method: sonication
chip antibody: FLAG M2 agarose affinity gel antibody SIGMA A2229
age: 8 days
objective time: ZT1
Growth protocol Seeds were surface-sterilised using ethanol or vapour-phase sterilisation (Clough & Bent, 1998) and stratified in respective growth media in the dark at 4˚C for 72 hours. Growth cabinets were maintained at 65% relative humidity and 170 µmol/m2/s light, unless specified otherwise, and as specified per experiment seedlings were grown at 20˚C with a 12-hour photoperiod or at 22˚C with either continuous light, a 16-hour long-day photoperiod or an 8-hour short-day photoperiod.
For ChIP-seq, A. thaliana seedlings were grown for 8 days on 1-mm-pore nylon mesh rafts placed on half-strength MS medium (pH 5.7) with 0.8% w/v agar (P1001, Duchefa) (‘MS agar’ hereafter) (22˚C long days).
Extracted molecule genomic DNA
Extraction protocol The seedling were grown for 10 days and treated as indicated. 3 g plant material for each treatment was fixed under vacuum for 20min in 1xPBS (10 mM PO43−, 137 mM NaCl, and 2.7 mM KCl) containing 1% Formaldehyde (F8775 SIGMA). The reaction was quenched by adding glycine to a final concentration of 62mM. Chromatin immunoprecipitation (ChIP) was performed as described (Jaeger et al), with the exception that 100μl of ANTI-FLAG® M2 Affinity Gel (A2220 Sigma) were used for immunoprecipitation.
Sequencing libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina, IP-202-1024) and prepared according to manufacturer instructions. DNA libraries were quantified using a Qubit fluorometer, and liibrary profiles were analysed using a TapeStation 2200 with High Sensitivity D1000 ScreenTapes (5067-5584, Agilent). Libraries were sequenced on a NextSeq-500 (Illumina; single end, 75bp reads) according to Illumina guidelines.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Data processing Genome_build: TAIR10
Adapters were trimmed off from raw reads with Trimmomatic with argument "ILLUMINACLIP:$FA_ADAPTER:6:30:10 LEADING:3 TRAILING:3 MINLEN:36 SLIDINGWINDOW:4:15". Raw reads were mapped to the genome "TAIR10" with Bowtie2 under argument:"--no-mixed --no-discordant --no-unal -k2". Any read that mapped to more than one genomic location was discarded. PCR duplicate reads were removed with Picard using default setting.
Genomic binding profile was quantified in RPKM (Reads Per Kilobase per Million mapped reads) using a bin-size of 10bp. "deeptools.bamCoverage" is used.
For each treated ChIP-Seq library, peaks were called against a chipseq input sample 195CS13 using MACS2 with argument "--keep-dup 1 -p 0.1".
Supplementary_files_format_and_content: *.narrowPeak: containing MACS2-called peaks. as described
Supplementary_files_format_and_content: * RPKM-normalised bigwig track at 10bp resolution
Submission date Feb 10, 2021
Last update date Feb 11, 2021
Contact name Philip Wigge
Organization name Cambridge University
Department Sainsbury Laboratory
Street address 47 Bateman street
City Cambridge
ZIP/Postal code CB2 1LR
Country United Kingdom
Platform ID GPL19580
Series (1)
GSE166543 Ribosomes act as cryosensors in plants
BioSample SAMN17855404
SRA SRX10067413

Supplementary file Size Download File type/resource
GSM5074265_data.198CS18.narrowPeak.gz 509.2 Kb (ftp)(http) NARROWPEAK 13.5 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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