title: GSM3903881: WT_3D7-G7-Input_R_rep1_ChIRP-seq; Plasmodium falciparum; OTHER
library_construction_protocol: Ring-stage (15 hr post invasion ±5 hr) synchronized 3D7-G7 and the PfRrp6-DD-1C line were used in ChIRP assay. Parasites culture (1x109 rings) were cross-linked as the ChIP arrays. After three washes with cold 1 x PBS, the parasite pellets were lysed with the swelling buffer (0.1M Tris pH 7.0, 10mM KOAc, 15mM MgOAc, 1% NP-40, 1mM DTT, protease inhibitor, and RNase inhibitor) at 4℃ for 10min with rotator. After centrifuge at 10,000 rpm for 10 min at 4 ℃, the parasites were resuspended in 150μl SDS Lysis Buffer ( 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0 ) for sonication. Chromatin was sheared into 200-500 bp fragments with Bioruptor (BioruptorTM UCD-200) of highest power for 4x4 min at 30 sec intervals. Chromatin is diluted in 2 times volume of hybridization buffer (500mM NaCl, 1%SDS, 100mM Tris 7.0, 10mM EDTA, 15% Formamide, add DTT, PMSF, P.I, and RNase inhibitor fresh). 100 pmol probes were added to each reaction, which was mixed by end to-end rotation at 37℃ for 4 hours. Streptavidin-magnetic T1 beads were washed three times in lysis buffer, blocked with 500ng/μl yeast total RNA and 1mg/ml BSA for 1 hour at 37℃, and washed three times again in lysis buffer before resuspended in its original volume. 100μl washed/blocked T1 beads were added per 100 pmol of probes, and the whole reaction was mixed for another 45min at 37℃. Beads : biotin -probes : RNA : chromatin products were captured by magnets (Invitrogen) and washed five times with 40x beads volume of wash buffer (2 x SSC, 0.5% SDS, add DTT and PMSF fresh). After last wash buffer was removed carefully with P-10 pipette, beads were resuspended in 3x original volume DNA elution buffer (50mM NaHCO3, 1%SDS, 200mM NaCl), and DNA was eluted with 100μg/ml RNase A and 0.1U/μl RNase H. RNase elution was carried out twice at 37 ℃ with end-to-end rotation. Then chromatin was reverse-crosslinked with  0.2U/μl protease K at 65℃ overnight. DNA was then extracted with equal volume of phenol : chloroform : isoamyl. Eluted DNA was subject to high-throughput sequencing. To prepare sequencing libraries, 10 ng of input DNA, 3-5ng IPed DNA were end repaired, extended with 3′ A-overhangs and ligated to barcoded NextFlex adapters (Bio Scientific). Libraries were amplified using an optimized KAPA protocol using KAPA HiFi HotStart ready mix (KAPA Biosystems) and the following PCR program: 98°C for 3 min followed by 14 cycles of 98°C for 20 sec, 65°C for 30 s; 68°C for 30s; finally extended 68°C for 5min. Amplified libraries were size-selected for 300-600 bp using 2% agarose gels. ChIP-seq libraries were sequenced on the HiSeq X Ten machine.
attributes.tag: GEO Accession
attributes.value: GSM3903881