Gaiix sequencer

A. veronii strain ML09-123, recovered from channel catfish (Ictalurus punctatus), was sequenced using barcoded Illumina libraries prepared using a Nextera DNA Sample Prep Kit (Epicentre, Madison, WI). Paired sequences were obtained from an Illumina GAIIx sequencer using 150 bp read length (Illumina, Inc., San Diego, CA) (4,911,312 reads resulting in 118X coverage). Reads were screened using the “trim sequences” option of CLC Workbench version 6.5.1. (CLC Bio). Adaptors were removed, low quality sequences were removed, and contig creation and de novo assembly were conducted using CLC Workbench and Sequencher version 5.4 (Gene Codes Corporation). For annotation, the draft genome was submitted to RAST [22 (link)] and NCBI`s Prokaryotic Genome Automatic Annotation Pipeline (PGAAP).

TFs were ligated into pT7-FLAG-4 vector (Sigma-Aldrich) for Flag-tagging and inducible expression. Plasmids were cloned into E. coli MG1655 strains and checked for kanamycin selection. Fidelity of the clones were validated through sequencing. Western blot verified production of inducible Flag-tagged TF using 1 mM IPTG. ChIP assays were performed by induction of strains in LB media starting at OD₆₀₀ 0.2 with 1 mM IPTG for 2 h. Cells were fixed with formaldehyde and glycine and sheared through sonication before immunoprecipitation with anti-FLAG monoclonal antibody. Further pull-down was done using agarose protein G beads. Reverse cross-linking of samples was performed by incubation with Proteinase K. DNA purification was carried out using DNA purification kit (Qiagen). Library preparation was done using standard Illumina TruSeq ChIP Sample Preparation protocols. ChIP replicate experiments presented here were performed by students as part of final projects for course BE605 in Biomedical Engineering at Boston University. Multiplexed sequencing was performed on an Illumina GAIIx Sequencer that generated single 50 bp reads. Total reads generated for the sequencing runs ranged from 3.5 – 22 million reads with an average of 10.62 million reads. ChIP-Seq control samples were wild-type strains with and without empty vectors subjected to the same immunoprecipitation protocol.

Four days after cutting stress, the edges of sorghum leaf strips that exhibited color changes in response to injury were collected. As controls, leaf strips immediately after cutting stress were also collected. To extract RNA, five biological replicates were collected, immediately frozen in liquid nitrogen, and mixed to minimize the effect of transcriptome unevenness among plants. RNA quality was calculated with a Bioanalyzer 2100 algorithm (Agilent Technologies, Palo Alto, CA, USA); high-quality (RNA integrity number >8) RNA was used. The protocol used for extraction of RNA and sequencing with an Illumina GAIIx sequencer (Illumina, San Diego, CA, USA) has been described previously (Mizuno et al., 2010 (link)). Reads were deposited in the DDBJ (DNA Data Bank of Japan) Sequence Read Archive (Accession No. DRA001265).

Poly-A RNA was isolated with oligo-dT-coupled beads from 20 μg total RNA of each sample and used for first strand cDNA synthesis with random hexamers and Superscript II reverse transcriptase (Invitrogen). Second strand cDNA was synthesized by DNA polymerase I (Invitrogen) and the double-stranded cDNA was end-repaired. A 3′ dA overhang was added and the double-strand product was ligated with Illumina adapters. The adapter-ligated sample was size-selected to ~200 bp fragments by electrophoresis. After 15 PCR cycles, libraries were sequenced using an Illumina GAIIx sequencer. Transcriptomic data that were generated in earlier studies (Gene Expression Omnibus accession number GSE3101256 (link)) also were analyzed. These data include Illumina RNA sequencing reads from: seven organs, 38 developmental stages and under various abiotic challenges, including extreme temperature, salinity and air exposure. Acquisition and analysis of RNA sequencing data are as described in other parts of this study and follow that described previously14 (link).

For each sample, RNA was extracted in triplicate from 0.2 g soil using a phenol–chloroform extraction protocol [57 ], modified from Griffiths et al. [58 (link)]. Extracted nucleic acids were passed through the Allprep DNA/RNA Mini Kit (Qiagen Sciences, Maryland, USA) to separate RNA from DNA. RNA was treated with DNase using an on-column DNase digestion. For community RNA-Seq, metatranscriptomic libraries were prepared directly from total RNA without rRNA removal using the TruSeq RNA Kit (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Metratranscriptomic libraries were sequenced on an Illumina GAIIX sequencer using 150 basepair (bp) paired-end sequencing at Lawrence Berkeley National Laboratory with an average of 9.5 million paired raw reads per sample. Sequences were deposited at NCBI under PRJNA692617.

RNA-Seq was performed following induction of Nac and CsiR using the same TF inducible E. coli strains used in ChIP-Seq as described above. Control experiments under identical conditions were also performed on WT E. coli. 50 mL of TF-inducible strains were induced with 1 mM IPTG for 2 h starting at OD₆₀₀ 0.2 in LB media. Total RNA extraction was performed using TRIzol® reagent (LifeTechnologies). Samples were subjected to 1-h DNAse digestion and purified using RNeasy spin columns (Qiagen). Samples were processed using Ribo-Zero rRNA removal kits and library preparation was done using NEB Next ultra-directional RNA library prep kit for Illumina. Multiplexed sequencing was performed on an Illumina GAIIx Sequencer that generated single 40 bp reads. Total coverage for the sequencing runs ranged from 8–14 million reads with an average of 10 million reads.