alias: GSM4766509
title: GSM4766509: Input rep2; Plasmodium yoelii yoelii; ChIP-Seq
library_construction_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.
identifiers.namespace: GEO
identifiers.id: GSM4766509
attributes.tag: GEO Accession
attributes.value: GSM4766509