title: GSM3903886: WT_3D7-G7_R_rep2_HiC-seq; Plasmodium falciparum; Hi-C
library_construction_protocol: Ring-stage (10 hr post invasion ±5 hr) synchronized 3D7-G7 lines was used in ChIRP assay. Parasites culture (1x109 rings) was harvested and cross-linked immediately with 1% paraformaldehyde with rotation for 10 min at 37℃, and quenched with 0.125 M glycine for 5 min on ice. The fixed culture was washed twice with cold 1xPBS, crosslinked parasite pellets were resuspended in 1 ml of ice-cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA-630, protease inhibitor cocktail). Parasite membranes were disrupted by passing the lysate through 30G needle 15 times using a syringe. Samples were spun at 4000 g for 5 min at 4 °C. Pellets were resuspended in 36μl of ice cold 10x NEBuffer 3.1 and 324μl pure water, samples were mixed with 38μl of 1% SDS to a final concentration of 0.1% and incubated at 65°C for 10 minutes. Samples were mixed with 43μl of 10% Triton X-100 to a final concentration 1%, then added 9.3 µl of 10x NEBuffer 3.1. 100U (2 µl of 50 units/µl) DpnII was added to digest the DNA overnight at 37°C on a rocking platform in a total volume of 434.3µl .  Day 2. The sample quality was assessed by running on 0.75% agarose gel, the 10 µl digested DNA should run as a smear between 200-300bp. The sample was incubated at 65˚C for 20 mins, then put it on ice until cooled to room temperature. 60 µl Biotin Fill-in MaseterMix (6 µl 10x NEBuffer 3.1, 2 µl 5 mM dCTP, 2 µl 5 mM dGTP, 2 µl 5 mM dTTP, 25 µl 0.4 mM biotin-14-dATP, 10 µl 5 U/µl DNA polymerase I Klenow, 13µl milliQ) and 50.7 µl 1x NEBuffer 3.1 were added to each tube. Samples were incubated at 37°C for 3h in Rocker and placed on ice immediately after incubation. Tubes were centrifuged for 5 min at 2500g at 4°C and the supernatant was discarded, then the samples were mixed with 500 µl ligation mix (50 µl 10x ligation buffer NEB, 50 µl 10% Triton X-100, 2.5 µl 20 mg/ml BSA, 2µl T4 DNA ligase NEB, 395.5 µl water) and incubated at 16°C for overnight.  Day 3. Each sample was incubated at 65°C for at least 2h by adding 25µl 20 mg/ml proteinase K, another 25µl 20 mg/ml proteinase K was added to each tube and samples were incubated at 65°C overnight.  Day 4. DNA was extracted with the an equal volume of phenol : chloroform : isoamyl alcohol (25:24:1), The aqueous phase was mixed with sodium chloride and glycogen to a final concentration of 200 mM and 25 µg/ml, respectively. DNA was precipitated by adding equal volume Isopropanol and incubated at -80°C for 2h. Precipitated DNA was pelleted by centrifugation at 16,000g for 15min at 4°C. Pellets was washed twice with cold 75% ethanol and resuspended in 100 µl TLE buffer (10 mM Tris-HCl, 0.1mM EDTA, dH2O pH 8.0). DNA was incubated for 30 min at 37°C by adding 1 µl RNAse A (1mg/ml). After ligation check on 0.75% agarose gel, 5 μg of Hi-C DNA sample was added to 5 μl 10x NEBuffer 2.1, 0.125 μl 10 mM dATP, 0.125 μl 10 mM dGTP and 15 Units T4 DNA polymerase (NEB) in a total volume of 50 μl  and incubated at 20°C for 4 h. Reaction was stopped at 75°C for 20 min. DNA was subsequently purified with TIAGEN Purification Kit (5 μg/column). DNA pellets were resuspended and pooled in a total of 200 μl water and divided into 2 tubes for sonication. The DNA was sheared to a size of 300-500 bp and run 1 μl with glycerol on 2% agarose gel. The Hi-C sample was purified with the AMpure XP mixture (Beckman Coulter Fullerton, CA) and eluted with 40 μl TLE buffer. The DNA ends were repaired by adding 6 μl 10x ligation buffer (NEB), 6 μl 2.5 mM dNTP mix, 6 μl ATP, 2 μl Enzyme mix (NEB) and incubated at 20°C for 45min, 75°C for 20min. The biotin tagged Hi-C DNA was bound to Dynabeads MyOne Streptavin C1 Beads (Invitrogen) as follows. 10 μl of resuspended Streptavidin beads were washed twice with 400 μl Tween Wash Buffer (TWB) (5 mM Tris-HCl pH8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween) by incubating for 3 minutes at RT with rotation. After this and for all subsequent incubations or washes of Streptavidin beads, the beads were reclaimed by holding against a magnetic particle concentrator (Invitrogen) for 1 minute and the supernatant was removed. These reclaimed beads were then resuspended in 400 μl 2x Binding Buffer (BB) (10 mM Tris- HCl pH8.0, 1 mM EDTA, 2 M NaCl) and combined with 390 μl Hi-C DNA(330 μl TLE). The mixture was incubated at RT for 15 minutes with rotation. The supernatant was removed and the DNA bound Streptavidin beads were resuspended in 400 μl 1x BB and transferred to a new tube. The beads were washed with 100 μl 1x TLE, transferred to a new tube before a final resuspension in 40 μl 1x TLE. An 'A' was added to the 3' ends of the end repaired DNA by addition of 5 μl 10x NEBuffer2.1, 2 μl 5 mM dATP and 3 μl Klenow (5 U/μl exo-) (NEB) . The reaction was incubated at 37°C for 30 minutes followed by 65°C for 20 minutes to inactivate Klenow (exo-). The reactions were cooled on ice and the beads were washed with 400 μl 1x ligation buffer and resuspended in 38.9 μl 1x ligation buffer. 6 μl of Illumina Paired End adapters (Illumina, San Diego, CA) were ligated to the Hi-C DNA for 2 hours at 22°C in the presence of 1x ligation buffer and 5 μl Quick ligase (Ambion, Austin, TX). The ligated Hi-C DNA was isolated by holding against the magnet and washed with 400 μl of 1x TWB to remove non-ligated Paired End adapters. The beads were resuspended in a further 400 μl 1x TWB and the mixture was transferred to a new tube and the Streptavidin beads were recovered. This wash step was repeated with 200 μl 1x BB and finally 20 μl 1x NEBuffer 2.1. The beads were resuspended in 5 μl 1x NEBuffer 2.1. Next, test PCR reactions were performed to determine the optimal PCR cycles needed to generate enough library for sequencing. 5 μl Streptavidin bead bound Hi-C library was mixed with 0.5 μl primers (12.5 nM), 15 μl KAPA HiFi 2x mix buffer and 9 μl dH2O in a total volume of 30 μl. The temperature profile was 120 s at 95°C followed by 10 cycles of 20 s at 95°C, 30 s at 62°C, 150 s at 65°C and a final 5min extension at 65°C. 5μl PCR reactions were run on a 2% gel after 10 cycles and the optimal cycle number was determined. The remain sample was PCR with the chosen cycles. The PCR product was purified by mixing with 1.5x volume Ampure beads (Beckman Coulter, Fullerton, CA). The mixture was held against a magnet to separate the PCR product bound to the Ampure beads and the supernatant was discarded. The Hi-C library bound Ampure beads were washed twice with 1 ml 70% ethanol while the tube remained against the magnet. After air-drying the beads, the DNA was eluted by resuspending the beads in 30 μl of TLE buffer. The tube was held against a magnet and the supernatant containing the purified PCR products was transferred to a new tube. The amount of DNA in the Hi-C library was quantified on the 2% agarose gel. 10 ng Hi-C product was digested with ClaI to estimate the portion of molecules with valid biotinylated junctions and incubated at 37°C for 30 min. The entire volume of the reaction was run on a 2% agarose gel. Finally, the Hi-C library was sequenced with Illumina paired end sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3903886