title: GSM2027613: Pf3D7-TRZGFP-input; Plasmodium falciparum; ChIP-Seq
library_construction_protocol: Synchronized 3D7/TRZGFP and 3D7/TRZHA schizonts at 5% parasitemia were cross-linked by incubation with a final concentration of 1% formaldehyde for 15 min at 37ºC. The cross-linking reaction was quenched by addition of 0.125M glycine (final concentration). Nuclei were isolated by releasing parasites from iRBCs using 0.05% saponin followed by lysis in CLB (20 mM Hepes, 10mM KCl, 1mM EDTA, 1mM EGTA, 0.65% NP-40, 1mM DTT supplemented with protease inhibitor). Nuclei were washed and snap-frozen in CLB supplemented with 50% glycerol. To obtain fragmented chromatin for ChIP, nuclei were resuspended in sonication buffer (50 mM Tris pH:8.0, 1% SDS, 10mM EDTA, supplemented with protease inhibitor) and sonicated for 20 cycles of 30 sec ON/30 sec OFF (setting high, BioruptorTM Next Gen, Diagenode). Fragment sizes ranged from 200-600 bp as determined by decrosslinking a 50μl aliquot and running the purified DNA on a 1.5% agarose gel.    ChIPs were performed by incubating ~500 ng (DNA-content) sonicated chromatin from 3D7/TRZGFP and 3D7/TRZHA overnight while rotating at 4°C with 1 μg mouse anti-GFP (Roche Diagnostics, #11814460001) or 1 μg rat anti-HA 3F10 (Roche Diagnostics, #12158167001), respectively, in incubation buffer (5% Triton-X-100, 750mM NaCl, 5mM EDTA, 2.5mM EGTA, 100mM Hepes pH 7.6) with 10 μl protA and 10 μl protG Dynabeads (Life Technologies, 10008D and 10009D). Beads were washed for 5 min at 4°C while rotating with 400 μl of the following wash buffers: 2x wash buffer 1 (0.1% SDS, 0.1% DOC, 1.0% Triton-X100, 1mM EDTA, 0.5mM EGTA, 20mM Hepes pH 7.6), 1x wash buffer 2 (0.1% SDS, 0.1% DOC, 1.0% Triton-X100, 500mM NaCl, 1mM EDTA, 0.5mM EGTA, 20 mM Hepes pH 7.6), 1x wash buffer 3 (250mM LiCl, 0.5% DOC, 0.5% NP-40, 1mM EDTA, 0.5mM EGTA, 20mM Hepes pH7.6), 2x wash buffer 4 (1mM EDTA, 0.5mM EGTA, 20 mM Hepes pH7.6). The immunoprecipitated chromatin was eluted in 200 μl elution buffer (1% SDS, 0.1M NaHCO3) while rotating for 20 min at RT and decrosslinked in decrosslinking buffer (1%SDS, 0.1M NaHCO3, 1M NaCl) in a 45°C shaking heat-block overnight. In parallel, 30 μl of sonicated input chromatin was decrosslinked under the same conditions. The DNA was purified over QIAquick PCR columns (Qiagen) and ten separate anti-HA or anti-GFP ChIPs were pooled over one column. For each line, the purified DNA from ten ChIPs or 5ng of the respective input sample was used for sequencing library preparation. Libraries were end-repaired followed by the addition of 3' A-overhangs and the ligation of NEXTFlex barcoded adapters (Bio Scientific, #514122). The libraries were then amplified using the KAPA HiFi HotStart ready-mix (KAPA Biosystems) under the following conditions: 98°C for 2 min; 4 cycles of 98°C for 20sec, and 62°C for 3min; 62°C for 5 min. Amplified libraries were gel size-selected for ~230-330 bp fragments on a 2% E-Gel SizeSelection agarose gel (Invitrogen, #G6610-02). The size-selected libraries were amplified for another 8 cycles with the KAPA HiFi HotStart ready-mix and adapter dimers were removed by purification using Agencourt AMPure XP beads (Beckman Coulter, #A63880).     The following NEXTFlex barcode adapters were used:  Pf3D7-TRZGFP-chip       CGATGT NEXTflex ChIP-seq 6 Barcodes 1  Pf3D7-TRZGFP-input      TGACCA NEXTflex ChIP-seq 6 Barcodes 2  Pf3D7-TRZha-chip         ACAGTG NEXTflex ChIP-seq 6 Barcodes 3  Pf3D7-TRZha-input       GCCAAT NEXTflex ChIP-seq 6 Barcodes 4