title: GSM4774607: AP2-G2_GFP_Gam_Rep1; Plasmodium falciparum; ChIP-Seq
library_construction_protocol: Crosslinked nuclei of PfA2-G2 GFP parasite was sheared in SDS lysis buffer (0.1% SDS, 10 mM Tris pH 8.1, 1 mM EDTA) to obtain chromatin of fragment size of 100-150bp using an M220 focused-ultrasonicator (Covaris Inc.) using the following settings: peak power 75W, 2% duty factor, 200 cycles per burst and total treatment time of 600 s. To immunoprecipitate sheared chromatin is diluted 1:10 with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 150 mM NaCl). To reduce the non-specific background chromatin solution is pre-cleared using 20ul/ml of magnetic beads A/G (Millipore 16-663) for 2 hours at 4oC with gentle agitation. Supernatant is collected and 50ul of the aliquot is removed as input. Input is control without the actual IP to get rid of the background noise. Rest of the chromatin was incubated overnight at 4oC with 1 μg of polyclonal anti-GFP antibody (Abcam ChIP grade, abcam 290) or, as control, the same amount of IgG. The immunoprecipitated antibody/chromatin complex was collected using A+G magnetic beads and is washed extensively with wash buffers for 5 min under agitation at 4oC. Finally the complex is eluted using 100 µL of fresh elution buffer (10µl 20% SDS, 20µl 1M NaHCO3, 170µl H2O). The input and ChIP samples were reverse cross-linked overnight at 45oC using 0.2M final concentration of NaCl. The sample is treated with RNAase (30min at 37oC) and proteinase K (3 µL of 20 mg/mL with 2 hours incubation at 45oC) and purified by using the QIAGEN MinElute PCR purification kit. 5-10 ng of ChIP enriched DNA was used as the starting material for preparing the Illumina TruSeq libraries. For accurate quantification of the ChIP DNA Qubit dsDNA HS assay was used. Barcoded TruSeq libraries for NextSeq (high output 150x150) single-end sequencing were constructed using NEBNext DNA library Prep reagents (New England Biolabs) by following the standard Illumina library preparation protocol. First ChIP DNA was end repaired for 30 minutes at 20oC and was cleaned using Ampure XP beads.  Then 3'ends are adenylated for 30 minutes at 37oC to prevent them ligating from one another and was cleaned again on beads. NEXTflex illumina DNA barcodes (diluted 1/10) were ligated to DNA fragments using NEB quick ligase incubating at 20oC for 10 minutes followed by the bead clean up steps. The resulting DNA is then size selected for 250bp inserts, PCR amplified and purified. The final library was quantified using Qubit fluorometer HD DNA kit and was analyzed using Agilent DNA 1000 Bioanalyzer to asses the quality, size distribution and detection of any artifacts.