GSM4276502: ChIP-seq_PfArp4_H2A.Z_input; Plasmodium falciparum; ChIP-Seq
In this experiment, highly synchronized infected red blood cells (iRBC) were collected and centrifuged to remove a portion of the supernatant. Parasites were extracted with 0.15% saponin and washed with pre-chilled PBS until the supernatant was clear. 2ml Lysis Buffer (containing 100× protease inhibitor and 100× DTT) was added to the sample, mixed well, and leave it on ice for 30min to lyse the body. The lysate was placed in a grinding tube and 50ul of 10% NP-40 was added. The cell debris was subjected to liquid phase ultrasound, after centrifuging the ultrasound product, took the supernatant and took 20ul of the supernatant as input (input was stored at -80°C). 100µl of salmon sperm DNA Agrose magnetic beads (50µl/ml) and protease inhibitor were added to the remaining supernatant, samples were incubated overnight. After overnight incubation, 4ug of anti-HA antibody and IgG antibody were added and incubated at 4°C for 2h. The buffers were added in the following order to wash the magnetic beads: the first step is to use 1ml of low-salt wash buffer, followed by the high-salt wash buffer, and finally add the LiCl wash buffer to the EP tube, the sample was incubated at 4°C for 5min, and let stand on ice for 2min. Samples were centrifuged at 1000rpm and 4°C for 1min, discard the supernatant after centrifugation. In the second step, 1ml of TE buffer was added to the EP tube and the EP tube was incubated at 37°C for 5min. Samples were centrifuged at 1000rpm and 37°C for 1min. Discard the supernatant and wash twice. The beads and Input (stored at -80°C) were eluted with 250µl of Elution Buffer, 10µl of 5M NaCl was added to the eluate, and samples were incubated at 65°C overnight. The DNA was extracted by phenol/chloroform and phenol/chloroform/isoamyl alcohol (25:24:1, pH7.8). DsDNA was then extracted by immunoprecipitation and decrosslinking. Take 14µl of RNase-free water to dissolve the extraction product. The dsDNA was extracted by immunoprecipitation and de-crosslinking, and the dsDNA stored at -80°C was taken out. The END-IT kit was used to repair the sticky ends to blunt ends. The reaction procedure is as follows: ChIP DNA 8ng, 10x NEB Next end repair buffer 1.5µl, dNTP mix 1.5µl, ATP 1.5µl, NEB NEXT end repair enzyme mix 0.5µl, dH2O up to 15µl. Incubate the reaction tube in a thermal cycler at 25°C for 45 minutes. Then add A-tailing to the blunt end of DNA. The reaction procedure is as follows: End repaired DNA 10µl, NEB Next A-tailing buffer 1.5µl, Klenow (3-5' exo-) 0.5µl, 2.5mM dATP 2µl, dH2O 1µl. Incubate the reaction tube in a thermal cycler at 37°C for 30 minutes. The third step is to connect the adapter to the DNA. The reaction procedure is as follows: End-Repaired A-tailed DNA 7µl, Indexed adaptor (diluted 1/10 in buffer EB) 1µl, NEB quick ligase 1µl, 10xT4 DNA ligase buffer 1µl. Incubate the reaction tube in a thermal cycler at 20°C for 10 minutes. The size of the DNA fragments was selected using Ampure beads. The required fragment size for the experiment is 300-600 bp. Finally, 10µl RNase-free water was used to dissolve the DNA fragment of the target size. The procedure for adaptor-ligated DNA amplification is as follows: DNA from previous step 10µl, 5x HF buffer 6µl, 10Mm dNTP 0.9µl, primer mix 0.5µl, KAPA HiFi enzyme 0.6µl, dH2O 12µl. Run program: 95°C 3min 1cycle, 98°C 20sec 14cycles, 65°C 30sec 14cycles, 65°C 30sec 14cycles, 65°C 3min 1cycle, 10°C 1cycle. The PCR products were cleaned up and eluted with 14µl EB, and the eluted products were sent for sequencing. The library were sequenced on an Illumina HiSeq Xten systerm with PE150 strategy.
GEO Accession
GSM4276502