Study of Capture sequencing of Tn5 transposase accessible chromatin DNA libraries in Arabidopsis thaliana. Ath_mutants_FANSATACCapSeq_20170630_NextSeq500_PE_MidOutput

Effector-triggered immunity (ETI) results in changes in gene transcript abundance. Previously, Jones Laboratory identified a list of ETI-specific early response genes (ERGs) at their transcript level through a time-course genome-wide RNAseq (Sohn et al. 2014 PLOS Genetics). We ask how the ERGs are regulated in ETI-specific manner, and what regulatory elements could be involved. Based on the literature, we selected several mutants (eds1-2[Col-0], rrs1-3 rrs1b-1, sard1 cbp60g, myc2 myc3 myc4, t) to test if the chromatin status ('open' or 'close', and transcription factor accessibilities) are directly associated with ERGs expressions upon ETI induction through Pf0-1 carrying AvrRps4 (ETI plus PAMP-triggered immunity [PTI]). The control plants are wild-type (WT) Col-0. The control treatments are untreated Col-0, mock treatment (10mM MgCl2) on all genotypes, and PTI alone (infiltration of Pf0-1 carrying AvrRps4 KRVY135-138AAAA mutant). We collected 2 leaves from each individual plant, and we collected 6 leaves from each genotype and each treatment at 4 hours post-infiltration. The OD600 of each Pf0-1 strains for infiltration is 0.2 in 10mM MgCl2. All plants were grown in a short-day controlled light chamber (JIC B5102). Every treatment and genotype have three biological replicates (3 batches of samples are collected on independent dates, and each batch contains one full set of samples from the WT and all selected mutants with all treatments. Leaves samples are directly ground in pre-cold nuclei isolation buffer (NIB). After 2M sucrose cushion in the NIB, nuclei pellets are resuspended in the resuspension buffer and stained with SYBR™ Green I Nucleic Acid Gel Stain, 10,000X concentrate in DMSO (ThermoFisher Scientific S7585) and sorted by the strength of GFP signal on Sony flow cytometry (cell sorter SH800). Approximately 1000 nuclei per sample are treated with Illumina Nextera Tn5 0.1uL enzyme in 5uL reaction mix at 37 degrees for half an hour. Tagmented nuclei samples are amplified by dual index primers designed by ourselves. Each library is barcoded with customer designed dual indexes (P5 and P7) with 9nt-index-nucleotide inserts. Post AMPure beads purification, the quality, and purity of the ATAC DNA libraries were tested through Bioanalyzer with Agilent DNA high sensitivity kit. Instead of genome-wide sequencing, we designed 120nt RNA bait libraries (http://www.mycroarray.com) to enrich the sequences from 52 genes of interests (ERGs and control genes) (capture sequencing or CapSeq). . Before the CapSeq, we mixed equal molar (pM) of each ATACseq libraries (only take the size between 200bp to 1000bp into the consideration of molar concentration) with different indexes. We spiked in 4 Col-0 genomic DNA (gDNA) libraries generated with Nextera kit as CapSeq control. The quantity of each gDNA library is 10-fold less than each cDNA library. Meantime, we also blend in sperm nuclei (3 replicates) and vegetative nuclei (3 replicates) FANSATAC samples from Dr. Xiaoqi Feng's lab. For the final mixture, our FANSATACCapSeq:'Xiaoqi's samples' molar ratio is 9:1, so we expect to see 90% reads are for our samples, 10% reads are for Xiaoqi's samples. Following the Standalone protocol from NextSeq, we used the 1.8pM final library as the input for sequencing. We used NextSeq® 500/550 Midi Output Kit v2 (150 cycles) (FC-404-2001) to perform the pair-end (PE) and dual-index RNAseq on the NextSeq 500 machine located in JIC.

效应子触发的免疫（Effector-triggered immunity, ETI）会导致基因转录本丰度发生改变。此前，琼斯实验室（Jones Laboratory）通过全基因组时间进程RNA测序（RNAseq）在转录水平上鉴定出了一系列效应子触发免疫特异性早期应答基因（ERGs）（Sohn等，2014，《公共科学图书馆·遗传学》（PLOS Genetics））。本研究旨在探究这些早期应答基因以效应子触发免疫特异性方式被调控的机制，以及可能参与其中的调控元件。基于已有文献，我们选取了若干突变体（eds1-2[Col-0]、rrs1-3 rrs1b-1、sard1 cbp60g、myc2 myc3 myc4、t），以测试在通过携带AvrRps4的Pf0-1菌株诱导效应子触发的免疫（同时伴随病原相关分子模式触发的免疫（PAMP-triggered immunity, PTI））时，染色质状态（“开放”或“闭合”状态以及转录因子可及性）是否与早期应答基因的表达直接相关。对照植株为野生型（WT）Col-0，对照处理包括未处理的Col-0、对所有基因型植株进行的模拟处理（10mM氯化镁（MgCl2））以及仅诱导病原相关分子模式触发的免疫的处理（接种携带AvrRps4 KRVY135-138AAAA突变体的Pf0-1菌株）。我们在接种后4小时采集样本：每株植株采集2片叶片，每个基因型及每种处理组合共采集6片叶片。用于接种的各Pf0-1菌株的菌液浓度（OD600）为0.2，溶剂为10mM氯化镁溶液。所有植株均种植于短日照可控光照培养箱（JIC B5102）中。每种处理及每个基因型均设置3次生物学重复：在不同日期分3批采集样本，每批包含野生型及所有选取的突变体在所有处理下的完整样本组。叶片样本直接置于预冷的细胞核分离缓冲液（NIB）中进行研磨。经NIB中的2M蔗糖垫离心后，将细胞核沉淀重悬于重悬缓冲液中，并用SYBR™ Green I核酸凝胶染色剂（10,000X DMSO浓缩液，赛默飞世尔科技（ThermoFisher Scientific）货号S7585）进行染色，随后通过索尼流式细胞仪（细胞分选仪SH800）根据GFP信号强度进行分选。每个样本取约1000个细胞核，在5μL反应体系中加入0.1μL Illumina Nextera Tn5酶，于37℃孵育30分钟进行转座酶标记。转座酶标记后的细胞核样本通过我们自主设计的双端索引引物进行扩增。每个文库均通过定制的双端索引（P5和P7）进行条形码标记，索引序列长度为9个核苷酸。经AMPure磁珠纯化后，使用安捷伦高灵敏度DNA试剂盒通过生物分析仪检测转座酶可及性测序（ATACseq）文库的质量与纯度。本研究未采用全基因组测序策略，而是设计了长度为120nt的RNA诱饵文库（http://www.mycroarray.com），以富集52个目标基因（包括早期应答基因及对照基因）的序列（即捕获测序（CapSeq））。在进行捕获测序前，我们将不同索引标记的ATACseq文库按等摩尔浓度（pM）混合，摩尔浓度计算仅考虑200bp至1000bp区间的文库片段。我们加入4份使用Nextera试剂盒构建的Col-0基因组DNA（gDNA）文库作为捕获测序的对照，每份基因组DNA文库的投入量为每份cDNA文库的1/10。同时，我们还混入了来自冯晓琦博士实验室的3份生物学重复的精子细胞核FANSATAC样本与3份生物学重复的营养细胞核FANSATAC样本。最终混合体系中，本研究的FANSATACCapSeq样本与冯晓琦博士实验室样本的摩尔比为9:1，因此预期测序reads中90%来自本研究样本，10%来自冯晓琦博士实验室样本。按照Illumina NextSeq的独立测序流程，我们以终浓度为1.8pM的混合文库作为测序投入量。我们使用NextSeq® 500/550中通量输出试剂盒v2（150个循环，货号FC-404-2001），在约翰英纳斯中心的NextSeq 500测序仪上进行双端测序（PE）及双端索引RNA测序。