ena-STUDY-JIC-20-09-2019-16:28:20:389-187
Isolation and sequencing of the mRNAs/cDNAs from wild-type and selected mutant Arabidopsis lines for studying the roles of corresponding genes in ETI early response gene regulations.
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, tpr1 tpl tpr4, pad4 ein2 dde2 sid2, sid2) to test the ERGs expressions upon ETI induction through Pf0-1 carrying AvrRps4 or AvrRpt2 (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 or leaf discs from each individual plant, and we collected 6 leaves or leaf discs from each genotype and each treatment at 4 hours post-infiltration. The OD600 of each Pf0-1 strains for infiltration is 0.2 in 10MgCl2. 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). Total RNAs are isolated with TriReagent (Sigma T9424) and Zymo RNA Clean and Concentrator kit. mRNAs are isolated with Dynabeads™ Oligo(dT)25 (ThermoFisher Scientific 61002) by purifying with the beads twice. Total RNAs and mRNAs quality and purity were tested through Bioanalyzer with Agilent RNA 6000 Nano and Pico kit, respectively. The first strands of cDNAs are synthesized with random decamers (Invitrogen AM5722G) and SuperScript™ III Reverse Transcriptase (Invitrogen 18080044 ). The second strands of cDNAs are followed the same protocol reported in the literature (Rallapalli et al 2014 BMC Genomics). cDNA libraries are generated through Illumina Nextera kit with 100pg input for each sample. 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). Each library is barcoded with customer designed dual indexes (P5 and P7) with 9nt-index-nucleotide inserts. Before the CapSeq, we mixed equal weight (ng) of each RNAseq libraries with different indexes, because they have very similar size distribution on the Bioanalyzer. 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. Following the Standalone protocol from NextSeq, we used 1.8pM final library as the input for sequencing. We used NextSeq® 500/550 High Output Kit v2 (75 cycles) (FC-404-2005) to perform the single-end (SE) and dual-index RNAseq on the NextSeq 500 machin 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, tpr1 tpl tpr4, pad4 ein2 dde2 sid2, sid2) to test the ERGs expressions upon ETI induction through Pf0-1 carrying AvrRps4 or AvrRpt2 (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 or leaf discs from each individual plant, and we collected 6 leaves or leaf discs from each genotype and each treatment at 4 hours post-infiltration. The OD600 of each Pf0-1 strains for infiltration is 0.2 in 10MgCl2. 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). Total RNAs are isolated with TriReagent (Sigma T9424) and Zymo RNA Clean and Concentrator kit. mRNAs are isolated with Dynabeads™ Oligo(dT)25 (ThermoFisher Scientific 61002) by purifying with the beads twice. Total RNAs and mRNAs quality and purity were tested through Bioanalyzer with Agilent RNA 6000 Nano and Pico kit, respectively. The first strands of cDNAs are synthesized with random decamers (Invitrogen AM5722G) and SuperScript™ III Reverse Transcriptase (Invitrogen 18080044 ). The second strands of cDNAs are followed the same protocol reported in the literature (Rallapalli et al 2014 BMC Genomics). cDNA libraries are generated through Illumina Nextera kit with 100pg input for each sample. 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). Each library is barcoded with customer designed dual indexes (P5 and P7) with 9nt-index-nucleotide inserts. Before the CapSeq, we mixed equal weight (ng) of each RNAseq libraries with different indexes, because they have very similar size distribution on the Bioanalyzer. 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. Following the Standalone protocol from NextSeq, we used 1.8pM final library as the input for sequencing. We used NextSeq® 500/550 High Output Kit v2 (75 cycles) (FC-404-2005) to perform the single-end (SE) and dual-index RNAseq on the NextSeq 500 machin located in JIC.