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Sample GSM4979247

Query DataSets for GSM4979247

Status

Public on Apr 20, 2021

Title

Input for post-stage II gametocytes_rep2

Sample type

SRA

Source name

Post-stage II gametocytes

Organism

Plasmodium falciparum NF54

Characteristics

strain: NF54
day of development: Day 7
parasite/gametocyte stage: Stage III, Stage IV gametocytes
chip antibody: None

Treatment protocol

Chromatin was cross-linked using 1% formaldehyde (10 min, 37°C) followed by 0.125 mM glycine quenching prior to extraction. Gametocytes were then isolated from host erythrocytes using 0.1% (w/v) saponin (10 min, 4°C)

Growth protocol

Gametocyte cultures were generated as described previously (Reader et al., 2015, DOI: 10.1186/s12936-1015-0718-z) P. falciparum (NF54) asexual parasites [maintained in human erythrocytes (5% haematocrit) in complete culture medium (RPMI 1640 (Sigma-Aldrich) supplemented with 25 mM HEPES, 20 mM glucose, 200 μM hypoxanthine, 0.2% (w/v) sodium bicarbonate, 24 μg/ml gentamycin and 0.5% (w/v) AlbuMAX II) at 37°C and under hypoxic conditions (90% N2, 5% O2, and 5% CO2)] were synchronised (>95% ring stage parasites) using 5% D-sorbitol for two consecutive replication cycles and were then subjected to glucose starvation and a reduced haematocrit (4%) to induce gametocyte commitment. N-acetylglucosamine (50 mM) supplemented media was changed daily from day 1 of development to remove residual asexual parasites.

Extracted molecule

genomic DNA

Extraction protocol

ChIP was performed as previously described (Josling et al., 2020, DOI: 10.1038/s41467-020-15026-0) Nuclei were isolated on day 2 (Pre-stage II gametocytes), day 7 (stage II gametocytes), or day 7 (Post-stage II gametocytes) of gametocyte development. Crosslinked nuclei were resuspended in Covaris shearing buffer (containing 0.1% SDS, 10 mM Tris pH 8.1, 1 mM EDTA) followed by sonication using a Covaris M220 ultrasonicator (sonication conditions: 5% duty cycle, 75 W peak incident power, 200 cycles per burst, time 300 s). Sonicated chromatin was pre-cleared using Protein A/G magnetic beads (Pierce) for two hr, followed by incubation of the pre-cleared chromatin with antibodies and magnetic beads overnight. A small volume of the chromatin was treated as a mock immunoprecipitation (no antibody) to serve as input material. Anti-rabbit H3K36me2 (Abcam, ab9049) and H3K36me3 (abcam, ab9050) antibodies were used for immunoprecipitations with beads washed and bound to DNA the following day using elution buffer. Reversal of crosslinking was preformed overnight at 45°C followed by the treatment of DNA with RNase A (37°, 30 min) and Proteinase K (45°, 2hr). The DNA was purified using the Qiagen MinElute kit and quantified by Qubit HS DNA assay.
Barcoded libraries were constructed as previously described (Josling et al., 2020, DOI: 10.1038/s41467-020-15026-0) using NEBNext DNA library Prep reagents (New England Biolabs) following a standard preparation protocol for Illumina TruSeq single-end sequencing. DNA was end-repaired (30 min, 20°C), purified (Agencourt AMPure XP beads, Beckman Coulter), dA-tailed (30 min, 37°C) and purified once more using Ampure XP beads. Illumina DNA barcodes (NEXTflex, 1/10 dilution) were ligated to DNA fragments with T4 DNA ligase (15 min, 20°C) with DNA subsequently size selected for 250 bp inserts (Ampure XP beads) and PCR-amplified (12-15 cycles) with Kapa HiFi (Kapa biosystems).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

Post-stage II input_2

Data processing

Sequencing reads were trimmed using Trimmomatic (v 0.32.3)
Trimmed reads were mapped to the P. falciparum 3D7 genome (v39, obtained from PlasmoDB) using BWA-MEM (v0.4.1)
Samtools (v1.1.2) was used to remove duplicate reads
bamCoverage, bamCompare and bigwigCompare (deepTools, v3.1.2.0.0) tools were used to obtain log2-transformed ChIP/input tracks, averaged for two biological replicates for further analysis
deepTools (v3.1.2.0.0) bamCoverage and htseq-count were used to obtain read counts, with the P. falciparum 3D7 genome annotation (v39) obtained from PlasmoDB. These counts were used to calculate average log2ChIP/input values and averaged for two independent biological replicates for each population except H3K36me3 in pre-stage II gametocytes (only 1 replicate). These values were used for subsequent analysis; average log2ChIP/input values for 750 bp upstream of start sites are provided for each gene within each sample as a processed data file.
Genome_build: P. falciparum 3D7 v39
Supplementary_files_format_and_content: Processes data files: bigwig files obtained using bamCoverage, bamCompare and bigwigCompare from deepTools (v3.1.2.0.0) representing log2 -transformed ChIP/input averaged for two biological replicates for each sample (Except for H3K36me3 in pre-stage II with only 1 replicate).
Supplementary_files_format_and_content: Upstream_read_count_matrix contains average log2ChIP/input scores for 750 bp upstream of start sites for all P. falciparum genes for which sequencing data was obtained for pre-stage II, stage II and post-stage II gametocyte samples.

Submission date

Dec 17, 2020

Last update date

Apr 20, 2021

Contact name

Jessica I Connacher

E-mail(s)

[email protected]

Phone

0718803047

Organization name

University of Pretoria