title: GSM4979241: Input for stage II gametocytes_rep2; Plasmodium falciparum NF54; ChIP-Seq
library_construction_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).
attributes.tag: GEO Accession
attributes.value: GSM4979241