GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2947036

Query DataSets for GSM2947036

Status

Public on Jul 06, 2018

Title

gDNA_ATACseq

Sample type

SRA

Source name

gDNA_ATACseq

Organism

Plasmodium falciparum

Characteristics

subclone: 10G
cell type: culture of parasite cells
developmental stage: schizont (40-45h)

Growth protocol

Parasites were cultured in B+ erythrocytes (3% hematocrit) under standard conditions with media containing Albumax II and no human serum.

Extracted molecule

genomic DNA

Extraction protocol

Cultures were tightly synchronized to a defined 5 h age window by purification of parasites at the schizont stage using Percoll gradients (63% Percoll) followed by sorbitol lysis 5 h later, which eliminates erythrocytes infected with late asexual stages (trophozoites and schizonts). Parasites were collected at the following times post invasion: 10-15 hpi (early ring stage), 20-25 hpi (late ring stage), 30-35 hpi (trophozoite stage) and 40-45 hpi (schizont stage). Genomic DNA was isolated by proteinase K treatment (0.2 mg/ml, 55°C for 3 hr and 95°C for 25 min), then RNase treatment (1.25 mg/ml, 37°C for 30 min). Genomic DNA was purified from schizonts by phenol/chloroform/isoamyl alcohol (25:24:1, pH 8.0) extraction, with an additional chloroform extraction, and precipitated with ethanol and NaOAc (3mM, pH 5.5), and resuspended in TE (10mM Tris-HCl pH7.5, 1mM EDTA). 10ng of gDNA used for a standard ATAC-seq protocol as described by the Nextera library preparation protocol by Illumina (5 min at 55º C).
ATAC-seq protocol was performed using 10 million cells for early ring, late ring and throphozoite cell stages and 1 million cells for schizonts that were obtained after saponin RBC lysis. Parasite cells were resuspended in lysis buffer in order to permeabilize membranes. The nuclei pellet were immediately resuspended in the transposition reaction mix (25 μl of 2x TD Buffer, 1.25 μl of Tn5 Transposase and 23.75 μl of nuclease-free water), and incubated for 30 min at 37ºC. The samples were purified using the Qiagen MiniElute Kit. Following purification, library amplification was carried out with 2X KAPA HiFi mix and 1.25 μM of Nextera primers. Optimal cycle number was determined using qPCR. ATAC-seq libraries were sequenced using an Illumina HiSeq2000 sequencer to obtain 20-40M of 2x50 bp paired-end sequencing reads.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

gDNA
10G and 1.2B are subclones derived from the 3D7 strain (Rovira-Graells et al. 2012. PMID 22415456).

Data processing

Paired reads were aligned to the P. falciparum reference genome Pf3D7-28 (PlasmoDB) with Bowtie2 (v.2.3.1), using parameters --no-unal --no-mixed -X 2000. Reads were trimmed 10 bases from each read 3' end (-3 10).
Alignment files were sorted and deduplicated using Samtools (v.1.4). Quality threshold of 10 in MAPQ score was applied.
To adjust for fragment size and make sure Tn5 cutting site was mapped, aligned reads were shifted +4bp for + strands and -5 bp for – strands.
Nucleosome-free fragments were extracted filtering mapped reads with a size threshold of 130 bp.
Peak calling on nucleosome-free reads was performed using MACS2 (v.2.1.1) using the module “callpeak” with the c- option where the input corresponds to the transposed genomic DNA (gDNA) that we use as a control. Parameters used were as default except for -g 2.41e7 --keep-dup all -q 0.01 --nomodel --shift -35 --extsize 75 and -B.
We performed independent peak calling for each biological replicate, on the pooled sample and on the pseudoreplicates, and keep only consensus THSSs regions if present in the three data sets (replicate, pooled and pseudoreplicate). THSSs regions located in mitochondrion and apicoplast chromosomes were removed.
THSSs regions were annotated to genomic features using HOMER and the ones corresponding to the same genomic region from different stages merged using bedtools merge.
Genome_build: Pf3D7-28 (PlasmoDB)
Supplementary_files_format_and_content: *_bdgcmp-ppois.bedgraph: Tracks of ATAC signal, normalized and input (gDNA) corrected, per stage of development. The bedGraphs were built with MACS2 “bdgcmp" module (-m ppois).
Supplementary_files_format_and_content: *_MACS2_peaks_merged_ATAC_rpkm_counts.txt: Counts of normalized (RPKM) and input corrected nucleosome-free reads per stage of development and subclone computed for MACS2 merged peaks (txt).
Supplementary_files_format_and_content: *_track.bed: Peaks

Submission date

Jan 24, 2018

Last update date

Jul 06, 2018

Contact name

Jose Luis Ruiz Rodriguez

Organization name

IPBLN-CSIC

Street address

Avda. del Conocimiento 17. P. T. Ciencias de la Salud

City

Granada