Microplex library preparation kit

Chromatin from synchronous-rings-stage parasites of 3D7 clone G7 was prepared and 3*10⁸ cells per ChIP used for the previously described protocol (Lopez-Rubio et al., 2013 (link)). Briefly, chromatin was crosslinked in 1% formaldehyde for 10 min (Sigma-Aldrich, #SZBD1830V), sheared to an average length of 300 bp using the BioRuptor Pico, and individual histone modifications were pulled down using 0.5 μg of antibody for H3K4me3 (Diagenode, cat # K2921004), H3K9me3 (Millipore, cat # 257833), and homemade rabbit polyclonal anti-PfHP1. 5 μl rabbit polyclonal anti-H4K31me1 and 15 μl anti-H4K31ac were used for each experiment. To generate Illumina-compatible sequencing libraries, the immunoprecipitated DNA and input was processed using the MicroPlex Library Preparation Kit (Diagenode C05010014) according to manufacturer’s instructions. The optimized library amplification step used KAPA Biosystems HIFI polymerase (KAPA Biosystems KK2101). Pooled, multiplexed libraries were sequenced on an Illumina NextSeq 500/550 system as a 150-nucleotide single-end run. The raw data were demultiplexed using bcl2fastq2 (Illumina) and converted to fastq format files for downstream analysis. Two biological replicates were analyzed for each antibody.

For each ChIP-seq reaction, ~ 10,000–15,000 mini-INTACT isolated bead-bound nuclei were processed using Low Cell # ChIP kit (Diagenode C01010070) as per manufacturer’s instructions. In brief, nuclei were fixed in 1% formaldehyde for 2 min, immediately quenched with glycine and then lysed using nuclear lysis buffer with protease inhibitor cocktail at room temperature for 5 min. PBS was added to dilute the lysate-bead mix and loaded in AFA tubes (Covaris Inc. 520045) for sonication. Ultra-sonicator (Covaris Inc. E220) was used to sheer chromatin to ~ 200 bp length, and chromatin was recovered from the supernatant after magnetic separation. ChIP was performed using the following ChIP-seq grade antibodies: H3K4me3 (Diagenode C15410003-50, Lot A5051-001P), H3K9me3 (Diagenode C15410193, Lot A1671-001P), H3K9/K14 ac (Diagenode C15410200, Lot A1756D), H3K27me3 (Diagenode C15410195, Lot A1811-001P), H3K27ac (Diagenode C154410196, Lot A1723-0041D), and H3K36me3 (Diagenode C15410192, Lot A1895P). Two biological replicates were performed for each histone mark, and input DNA was used as the control. Libraries for sequencing were prepared using MicroPlex Library Preparation kit (Diagenode C05010012) as per manufacturer’s instruction. Single-end 60 bp sequencing reads were obtained using Illumina Hi-seq 2500.

Targeted genomic regions were identified by using the cross-linking (X)-ChIP protocol as described previously^{40 (link)}. After the MV infection, cells were fixed 10 min with 1% formaldehyde and then quenched with glycine (125 mM). Chromatin was incubated overnight at 4 °C with Chip grade H3K4me3 antibody (5 μg, ab8580, Abcam) and equal amounts of Protein A&G Agarose Beads (Life technologies). Library preparation for sequencing was done following the instructions of TruSeq DNA sample preparation kit from Illumina or MicroPlex Library preparation kit from Diagenode.