GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5006003

Query DataSets for GSM5006003

Status

Public on Jan 14, 2021

Title

ATAC_seq_Meristems_rep3

Sample type

SRA

Source name

anantha inflorescences

Organism

Solanum lycopersicum

Characteristics

genotype: anantha-e1546 mutant in M82 background
tissue: inflorescences

Growth protocol

Tomato anantha-e1546 (Solyc02g081670) in M82 background seeds were directly sown into soil in 96-cell plastic flats and grown in a greenhouse under long-day conditions (16-hr light/8-hr dark) supplemented with artificial light from high-pressure sodium bulbs (∼250 μmol m−2 s−1). The temperature ranged between 26-28°C during the day to 18-20°C during the night, with a relative humidity of 40-60%. One-month old seedlings were transplanted to a 4L pot and grown in the greenhouse at the same conditions.

Extracted molecule

genomic DNA

Extraction protocol

Six biological replicates of 1g of freshly collected anantha inflorescences (meristematic enriched tissue) were ground in ice-cold isolation buffer (300 mM sucrose, 20 mM Tris pH 8.0, 5 mM MgCl2, 5 mM KCl, 5 mM beta-mercaptoethanol, 35% glycerol, 0.2% Triton X-100). After grinding, the suspension was filtered through a series of cell strainers (the smallest being 70 mm). Samples were further disrupted using a Dounce homogenizer, and then were washed four times with ice-cold isolation buffer. The enriched nuclei samples were resuspended in Resuspension Buffer (50 mM Tris pH 8.0, 5 mM MgCl2, 5 mM beta-mercaptoethanol, 20% glycerol).
For the transposase reaction 100,000 nuclei were resuspended in 22.5 ml freezing buffer (5 mM MgCl2, 0.1 mM EDTA, 50 mM TRIS pH 8.0, 40% glycerol), and were mixed with 2X DMF Buffer (66 mM Tris-acetate (pH 7.8), 132 K-Acetate, 20 mM Mg-Acetate, 32% DMF) and 2.5ul Transposase enzyme from Illumina Nextera Kit (Catalog No. FC-121-1031), and incubated at 37°C for 30 min. Tagmented DNA was isolated using NEB Monarch PCR Purification kit (Catalog No. NEB #T1030). Next-generation sequencing (NGS) libraries were amplified in two steps (Buenrostro et al., 2015) using KAPA HiFi HotStart ReadyMix PCR kit (Catalog No. KK2601). NGS libraries were PE50 sequenced on an Illumina MiSeq SY-410-1003 instrument.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina MiSeq

Data processing

ATAC-seq reads were trimmed with trimmomatic and aligned to Heinz SL4.0 reference genome with BWA mem. Reads that mapped to either chloroplast or mitochondria were filtered out together with PCR duplicate reads (picard MarkDuplicates) and multiple position aligned reads. Finally, only high quality properly paired reads were retained for the analysis (samtools view -b -h -f 3 -F 4 -F 8 -F 256 -F 1024 -F 2048 -q 30).
For Jbrowse visualization of the ATAC-seq, all six BAM files were merged anf coverage was calculated with genomeCoverageBed followed by bedGraphToBigWig command.
ATAC-seq peak calling was done by Genrich V0.6 (https://github.com/jsh58/Genrich) using all six libraries with the following parameters: -j -r -v -q 0.5 -g 30.
Genome_build: Tomato SL4.0 ITAG4.0
Supplementary_files_format_and_content: ATAC_seq_Meristem_Genrich_Rep_q_0.5_g30_Final.narrowPeak_sorted.bed - This file contain the ATAC-seq peaks called by Genrichusing all six libraries to calculate q-value. ATAC_seq_meristem_HQ_merged_final.bw - ATAC-seq reads genome coverage (merged).

Submission date

Jan 05, 2021

Last update date

Jan 14, 2021

Contact name

Zachary Lippman

E-mail(s)

lippman@cshl.edu

Phone

5163675035

Organization name

Cold Spring Harbor Laboratory