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| Status |
Public on Jan 26, 2021 |
| Title |
HA-IP rep1 |
| Sample type |
SRA |
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| Source name |
gametocytes
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| Organism |
Plasmodium yoelii yoelii |
| Characteristics |
strain: Plasmodium yoelii yoelii 17XNL
chip antibody: HA-Tag (C29F4) Rabbit mAb #3724
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mice infected with parasite were treated with sulfadiazine in drinking water for 24-32 hours to kill asexual stage parasites. Blood with high gametocytemia was collected from mouse orbital sinus into heparin tubes and depleted of leukocytes using NWF Filter (ZhiXing Bio, China). Gametocytes were immediately fixed with 1% of methanol-free formaldehyde (Pierce, #28906) at room temperature for 10 min with gentle shaking for crosslinking DNA-proteins. Fixed cells were subjected to lysis in 0.84% NH4Cl for 10 min on ice and then washed twice with gametocyte maintenance buffer (GMB, containing 137 mM sodium chloride, 4 mM potassium chloride, 1 mM calcium chloride, 20 mM HEPES, 20 mM Glucose, 4 mM sodium bicarbonate, 0.1% w/v bovine serum albumin, pH 7.25). The gametocytes were further enriched in a 60% Nycodenz gradient centrifugation and harvested for ChIP.ChIP assays were performed using SimpleChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technology, #56383). The crosslinked cells were lysed and followed by chromatin-shearing using Covaris M220 focused ultrasonicator (Covaris, Inc) with the procedures: peak power 75W, 20% duty factor, 200 cycles per burst and total treatment time of 600 sec. The distribution and concentration of sheared chromatin were detected by agarose gel electrophoresis and Nanodrop 2000 (Thermo Fisher). 10 μg of chromatin was immunoprecipitated with anti-HA rabbit antibodies (Cell Signaling Technology, AB_1549585). As a control, the same amount of IgG antibody (Cell Signaling Technology, AB_1031062) was used. Immunoprecipitated chromatin collected with A+G magnetic beads was extensively washed and eluted with elution buffer. The input and ChIP samples were reverse cross-linked overnight at 65°C in the presence of Proteinase K (Thermo Fisher, #AM2546) and purified using the QIAquick Gel Extraction Kit (QIAGEN, #28704).
ChIP samples were quantified using a Qubit 2.0 Fluorometer (Invitrogen, USA) and qualified by Agilent Bioanalyzer 2100 (Agilent Technologies, USA). For each sample, At least 5 ng ChIP product was used for library preparation using VAHTS Universal Pro DNA Library Prep Kit (Vazyme, #ND-608). The ChIP product was treated with End Prep Enzyme Mix for end repairing, 5’ Phosphorylation, and dA-tailing in one reaction, followed by ligation to adaptors with a “T” base overhang. Adaptor-ligated DNA was recovered using AxyPrep Mag PCR Clean-up (Axygen, #MAG-PCR-CL-50) and amplified by PCR for 10 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flowcell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. PCR products were cleaned using AxyPrep Mag PCR Clean-up, validated using an Agilent 2100 Bioanalyzer, and quantified by Qubit 2.0 Fluorometer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Basecalls performed using Bcl2fastq (v2.17.1.14)
pass filter data of fastq format were processed by Cutadapt (version 1.9.1) to obtain high quality clean data
The clean data were aligned with reference genome via software Bowtie
peak-calling:The mapping data (immunoprecipitated and input) were analyzed with the MACS2 (V2)
Genome_build: The reference genome sequences and gene model annotation files of P. yoelii were downloaded from PlasmoDB 36.
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Sep 03, 2020 |
| Last update date |
Jan 26, 2021 |
| Contact name |
Zhenkui Li |
| E-mail(s) |
[email protected]
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| Organization name |
Xiamen University
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| Street address |
Xiangan south road
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| City |
Xiamen |
| State/province |
Fujian |
| ZIP/Postal code |
361102 |
| Country |
China |
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| Platform ID |
GPL29103 |
| Series (2) |
| GSE157454 |
Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes [ChIP-seq] |
| GSE157457 |
Malaria parasite transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes |
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| Relations |
| BioSample |
SAMN16055579 |
| SRA |
SRX9072452 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4766508_HA-IP_rep1.bw |
55.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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