ena-STUDY-JIC-18-10-2019-12:11:51:028-34
Study of Capture sequencing of Tn5 transposase accessible chromatin DNA libraries in Arabidopsis thaliana
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.
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.