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Sample GSM2563161

Query DataSets for GSM2563161

Status

Public on Jul 24, 2017

Title

Si_WL_rep_3

Sample type

SRA

Source name

Setaria italica seedlings

Organism

Setaria italica

Characteristics

tissue type: Whole Leaf
tissue source: Third and Fourth Leaves
developmental stage: 18 days

Treatment protocol

Bundle Sheath tissues were isolated by filtering after shearing leaves in a Waring blender.

Growth protocol

S. italica, S. bicolor and Z. mays were grown under controlled conditions at the University of Cambridge. A growth room was set on a 12h:12h day/night cycle; temperature, 28oC day/20oC night; illumination, 400µm; humidity, 60%; CO2, ambient air. Germination: Sorghum bicolor and Zea mays seeds were imbibed in dH2O for 48h; Setaria italica, seeds were germinated on wet filter paper at 30oC overnight in the dark. Growth medium: 3:1 M3 compost to medium vermiculite (v/v) mixture, with a thin covering of soil. Seedlings were hand watered. For B. distachyon plants were grown under controlled conditions at the Sainsbury Lab Cambridge, first under SD condition 14h/10h, light/dark for 2 weeks and then shifted to LD 20h/4h, light/dark, for 1 week and harvested at ZT20. Temperature was set at 20oC, humidity 65% and light intensity 350µMols.

Extracted molecule

genomic DNA

Extraction protocol

Small fragments of DNA (~50-600bp) were isolated using gel purification following phenol chloroform extraction of DNaseI treated nuclei.
Libraries were created using the Hyper Prep DNA Library preparation kit according to the manufactures instructions and indexed for pooling using NextFlex DNA barcoded adapters (Bioo Scientific, Austin TX US). Libraries were quantified on a Tapestation DNA 1000 Screen tape and by qPCR using an NGS Library Quantification Kit (KAPA Biosystems) on an AriaMx qPCR system (Agilent). Libraries were then normalised, pooled, diluted and denatured for sequencing on the NextSeq 500 (Illumina, Chesterford UK) according to the manufacturer’s instructions. PhiX control library (Illumina) was spiked into the main library pool at 10% for quality control purposes and to improve library diversity.

Library strategy

DNase-Hypersensitivity

Library source

genomic

Library selection

DNAse

Instrument model

Illumina NextSeq 500

Description

Harvested between 4-6 hours after beginning of light cycle, Steven J. Burgess and Ivan Reyna-Llorens
DHS_Si_WL.regionPeak.txt
DGF_Si_WL.bed
DGF_DE_Si_WL.bed

Data processing

DNaseI digested reads were aligned to reference genomes using Bowtie2 using the following configuration: --local -D 15 -R 2 -N 0 -L 20 -i S,1,0.75
DNaseI hypersensitive sites (DHS) were identified using MACS2 using the following configuration: -p 1e-1 --nomodel --extsize 150 --shift -75 --llocal 50000
The final set of DHS sequences was determined by selecting the optimal number of peaks called on the pooled replicates. This number is determined by the maximum number of consistent peaks between replicates, as determined spp with the following configuration: [peaks_1] [peaks] -1 0 F 0.02.
Digital genomic footprints (DGFs) were identified within DHS using wellington with the following parameters: -fdr 0.05
Differential DGFs were identified with DHS using wellington bootstrap with the following parameters: -fdr 0.05
Genome_build: Bdistachyon_283_assembly_v2.0; Sbicolor_255_v2.0; Sitalica_164_v2; Zmays_284_AGPv3.
Supplementary_files_format_and_content: DHS are in MACS2 output BED6+4 format, DGF are Wellington output (BED), DGFs annotated with TF motifs BED3+2.

Submission date

Apr 04, 2017

Last update date

May 15, 2019

Contact name

Steven James Burgess

E-mail(s)

[email protected]

Organization name

University of Cambridge

Department

Plant Sciences