title: GSM8771646: 3D7-BDP5-FKBP, Biotin, 24to32hpi, plusBiotin, Rapa, DiBioCUT&Tag, rep2; Plasmodium falciparum; OTHER
library_construction_protocol: For nuclei isolation, parasite cultures were lightly crosslinked with 0.1% formaldehyde (Sigma, F8775), incubating for 2 min at 37 °C while shaking. Crosslinking was stopped by addition of glycine to 0.125 M final concentration. Samples were handled on ice from here onwards. Cells were harvested by 440 g centrifugation for 8 min at 4 °C, and washed with ice-cold PBS. Centrifugation was repeated and the pellet was washed with PBS supplemented with 1x EDTA-free Protease Inhibitor (Roche, 04693132001). Parasites were extracted by adding saponin (0.05% total concentration) and incubating at room temperature (RT) for 10 min. Nuclei were isolated by carefully transferring the extracted parasite mixture on top of a 0.25 M to 0.1 M sucrose gradient in cell lysis buffer (10 mM Tris pH 8, 3 mM MgCl2, 0.2% NP-40, 1x EDTA free Protease inhibitor (Roche, 04693132001); 15 mL 0.25 M Sucrose and 17.5 mL 0.1M Sucrose for 50 mL tubes, 4 mL 0.25 M Sucrose and 6 mL 0.1 M Sucrose for 15 mL tubes) and centrifuging for 12 min, 3100 g, 4 °C with acceleration and deceleration set to 1 (Eppendorf 5910 Ri, Rotor S-4x400). Supernatant was removed and nuclei washed in cell lysis buffer (10 min, 3500 g, 4 °C; Heraeus Fresco 21). Nuclei were counted in an automatic hemocytometer (BioRad, TC10 Automated Cell Counter) and were directly used for CUT&Tag. Optionally, nuclei were washed with cell lysis buffer containing 20% Glycerol and nuclei pellet was snap frozen in liquid nitrogen prior to storage at -80°C. For parasite isolations, parasite cultures were harvested by 440 g centrifugation for 8 min at RT and pellet was resuspended in 1 mL of 0.15% Saponin/PBS per 10 mL culture and incubated for 5 min on ice. Samples were vortexed shortly every minute. Samples were washed three times with ice-cold PBS (3500 g centrifugation for 3 min at 4 °C; Heraeus Fresco 21). Intact parasites were counted in an automatic hemocytometer and were directly used for CUT&Tag. Optionally, parasites were washed with PBS containing 20% Glycerol and parasite pellets snap frozen in liquid nitrogen prior to storage at -80 °C. Nuclei or whole parasite were resuspended in CUT&Tag wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0,5 mM Spermidine, 1x EDTA-free protease inhibitor) containing 0.1% Triton X-100 and permeabilized for 10 min on ice. Nuclei / parasites were pelleted by centrifugation (3500 g, 10 min, 4 °C) and resuspended in CUT&Tag wash buffer. Concanavalin A beads (Bangs Laboratories, BP531) were activated by resuspending into 10 volumes of Bead Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2), washed once on a magnetic rack and resuspended in the starting volume of bead slurry. Purified nuclei or parasites were bound to 10 µL beads per reaction by incubating for 10 min rotating at RT. The supernatant was removed and 50 µL antibody buffer (CUT&Tag wash buffer; 2 mM EDTA, 0.1% BSA) with primary Antibody (0.25 µL polyclonal rabbit αHP1; 0.5 µg αH3K9me3, Abcam 8988; 0.5 µL αBiotin (Cell Signaling Technology #D5A7; for DiBioCUT&Tag); 0.25µg αGFP, Roche 11814460001; or 0.5 µg normal rabbit IgG (MERCK, #12-370)) was added and incubated nutating over night at 4 °C. Unbound primary antibody was removed by washing once with 100 µL CUT&Tag wash buffer and samples were then incubated with secondary antibody (1.2 µg guinea pig anti-rabbit antibody, Antibodies-Online ABIN101961; or 0.5 µg goat anti-mouse antibody, EpiCypher 13-0048; in 100 µL CUT&Tag wash buffer, 1:100 dilution) for 1h on RT, nutating. The nuclei or parasites were washed on a magnetic stand twice with 100 µL CUT&Tag wash buffer and once with 100 µL CUT&Tag 300 Wash Buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0,5 mM Spermidine, 1x EDTA-free protease inhibitor). For all following wash steps CUT&Tag 300 wash buffer was used in order to quench potential affinity of protA-Tn5 to accessible chromatin regions. 2.5 µL commercial proteinA/G-Tn5 fusion protein (CUTANA™ pAG-Tn5 for CUT&Tag, Epicypher, 15-1017) was added in 50 µL CUT&Tag 300 wash buffer and incubated nutating for 1 h. Unbound proteinA/G-Tn5 fusion protein was removed by thrice washing with 100 µL CUT&Tag 300 wash buffer. The supernatant was removed and the nuclei or parasites were resuspended in 200 µL freshly prepared tagmentation buffer (CUT&Tag 300 wash buffer, 10 mM MgCl2). To perform tagmentation, samples were incubated in a PCR thermocycler (BioRad, T100) at 37 °C for 1h. Tagmentation was stopped and nuclei or parasite lysis was facilitated by addition of 10 µL of 0.5M EDTA pH8, 3 µL of 10% SDS and 1 µL of 50 mg/mL proteinase K. Samples were briefly vortexed and then incubated at 55 °C for 1 h. DNA fragments were extracted utilizing the DNA Clean & Concentrator - 5 kit (Zymogen, D4014) following manufactures instructions. DNA was eluted from the column with 26 µL of prewarmed elution buffer and DNA concentrations were assessed with Qubit dsDNA High Sensitivity Assay kit (Invitrogen, Q33231). Libraries were generated using maximal 50 ng of extracted DNA from CUT&Tag experiments and tagged fragments were amplified with unique combinations of i5 and i7 barcoded primers, enabling tracing the DNA fragments originating from separate experiments in the sequencing data. PCRs were performed in a total reaction volume of 50 µL using Kapa HiFi polymerase (use non-hotstart version for gap filling; Roche, KK2102) in a thermocycler with the following program: 58 °C for 5 min, 62 °C for 5 min (gap filling), 98 °C for 2 min, 12-16 cycles of 98 °C for 20 second and 62 °C for 10 seconds, 62 °C for 1 min and hold at 4 °C. Post-PCR DNA cleanup was performed by adding 50 µL (1x volume) of AMPure XP bead slurry (Beckman Coulter, A63882) and incubating for 10 min at RT, washing twice with 80% EtOH on a magnetic rack, and eluting in 16.5 µL of 10 mM Tris-HCl pH 8 for 5 min at RT. DNA concentrations of libraries were assessed with Qubit dsDNA High Sensitivity Assay kit (Invitrogen, Q33231) and library fragment size distribution was accessed by microfluidic gel electrophoresis (Agilent 2100 Bioanalyser) with the corresponding High Sensitivity DNA Kit (Agilent, 5067-4626). Sequencing was performed using an Illumina NextSeq 2000 instrument for 3-10 M reads per CUT&Tag sample; 59bp paired-end reads were generated. (DiBio)CUT&Tag, ChIPseq
identifiers.namespace: GEO
identifiers.id: GSM8771646_r1