ATAC-seq of monocytes from patients before and after anti-malarial treatment. ATAC-seq of monocytes from patients before and after anti-malarial treatment

Patients received standardized coartem therapy. Briefly, each pill contains 20 mg of artemether and 120 mg of lumefantrine. The dose and total coarse of tablets is based on the patients body weight. Blood was drawn from patients prior to anti-malarial treatment (day 0) and after treatment (day42 for children and day 28 for adults). Peripheral Blood Mononuclear Cells were isolated from all patients. Briefly, for PBMC isolation, whole blood from was diluted 1:2 in sterile PBS and overlaid on Ficoll Paque-PLUS in 50 mL conical tubes and centrifuged 800xg for 15 minutes at 25°C with no brake. Resulting buffy coats were collected and washed twice in RPMI 1640 medium, and then cells were treated with Red Blood Cell Lysis Buffer for 3 minutes at room temperature. Cells were washed in RPMI and counted on a hemacytometer. Monocytes were purified from PBMC using a miltenyi Pan Monocyte Isolation negative selection kit according to the manufacturer's instructions. For each sample. 50,000 viable cells were pelleted and lysed with cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin and incubated in ice for 3 minutes. The lysaste was washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but NO NP40 or digitonin. Nuclei were pelleted at 500 RCF for 10 min at 4 degrees celcius. The supernatant was discarded and the pellet was resuspended in 50 uL of transposition mixture (25 ul 2x TD buffer, 2.5 ul transposase (100nM final), 16.5 ul PBS,0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O). The reaction was incubated for 30 minutes in a thermomixer with 1000 RPM mixing. Zymo DNA Clean and concentrator kit (cat# D4014) was used as a cleanup step. The DNA was amplified at 98 degrees C for 30 seconds, then 13 cycles of: 98 degrees C for 10 seconds, 63 degrees C for 30 seconds, 72 degrees C for 1 minute.The library was evaluated in an Agilent TapeStation system and the DNA concentration evaluated by Qubit. Sequencing was performed on Illumina Nextseq 500 with Buffer cartridge v2: Ref: 15057941, High output reagent cartridge v2: Ref: 15057934, NextSeq accessory Box v2: Ref: 15058251, and High output Flow Cell cartridge v2.5: Ref: 20022408

受试者接受标准化复方蒿甲醚（Coartem）治疗。简言之，每粒药片含20毫克蒿甲醚（artemether）与120毫克苯芴醇（lumefantrine）。给药剂量与总药片疗程基于受试者体重。分别于抗疟治疗前（第0天）及治疗结束后采集受试者血液：儿童采血时间为治疗后第42天，成人则为治疗后第28天。
从所有受试者体内分离外周血单个核细胞（Peripheral Blood Mononuclear Cells，PBMC）。具体而言，PBMC分离流程如下：将全血以1:2比例用无菌磷酸盐缓冲液（PBS）稀释后，转移至50 mL离心管中并铺于Ficoll-Paque PLUS分层液液面，于25℃、800×g条件下离心15分钟，离心过程不使用刹车。收集分层得到的棕黄层（buffy coat），用RPMI 1640培养基洗涤两次，随后使用红细胞裂解液（Red Blood Cell Lysis Buffer）于室温下处理细胞3分钟。再用RPMI培养基洗涤细胞，通过血细胞计数板（hemacytometer）进行细胞计数。
使用美天旎（Miltenyi）全单核细胞阴性分选试剂盒，按照制造商说明书从PBMC中纯化单核细胞。每份样本取50,000个活细胞离心沉降，使用含0.1% NP40、0.1% Tween-20及0.01%洋地黄皂苷（Digitonin）的低温ATAC重悬缓冲液（ATAC-Resuspension Buffer，RSB）重悬并裂解，于冰上孵育3分钟。用1 mL含0.1% Tween-20但不含NP40与洋地黄皂苷的低温ATAC-RSB洗涤裂解液。于4℃、500相对离心力（RCF）条件下离心10分钟以沉降细胞核。弃去上清液，将沉淀重悬于50 μL转座反应混合液中（配比为：25 μL 2×TD缓冲液、2.5 μL转座酶（终浓度100 nM）、16.5 μL PBS、0.5 μL 1%洋地黄皂苷、0.5 μL 10% Tween-20、5 μL 无菌水）。将反应体系置于恒温混匀仪中，以1000转/分钟转速振荡孵育30分钟。使用Zymo DNA清洁与浓缩试剂盒（货号D4014）完成纯化步骤。
对DNA进行扩增：98℃预变性30秒，随后进行13个循环反应，每个循环包括98℃变性10秒、63℃退火30秒、72℃延伸1分钟。使用安捷伦TapeStation系统对文库进行质检，并通过Qubit荧光定量仪检测DNA浓度。使用Illumina NextSeq 500测序平台进行测序，配套试剂包括：v2版缓冲液试剂盒（货号：15057941）、v2版高通量试剂试剂盒（货号：15057934）、v2版NextSeq配套试剂盒（货号：15058251）以及v2.5版高通量流动池试剂盒（货号：20022408）。