Rta v2

For whole-genome sequencing, DNA was extracted by phenol-chloroform extraction, followed by column cleanup using a NucleoSpin DNA Plant II kit (Macherey-Nagel). Clustering and DNA sequencing of wild-type, plasmid-cured, and ALE-derived P. putida S12 strains were performed using Illumina cBot and HiSeq 4000 systems (GenomeScan BV, The Netherlands). Image analysis, base calling, and quality checking were performed with the Illumina data analysis pipeline RTA v.2.7.7 and Bcl2fastq v.2.17. Sequencing reads were assembled according to the existing complete genome sequence (GenBank accession no. CP009974 and CP009975) in Geneious software (18 (link)). Variant calling was performed by aligning the reads from the ALE-derived strains to their corresponding parental strains.

RNA from sorted microglial cells was isolated using the miRNeasy Serum/Plasma Kit (QIAGEN; 217184) and quantified on the Agilent BioAnalyzer 2100. cDNA libraries were generated using SMARTSeq2 v4 and Nextera Low Input Library Prep Kit. Samples were multiplexed and sequenced on the Illumina HiSeq 4000.
RNA from frozen bulk tissue was isolated using Trizol and chloroform, followed by DNase and clean up using the Rneasy Kit (QIAGEN; 74106). Libraries were generated using the TruSeq Stranded mRNA Library Prep Kit. Samples were multiplexed and sequenced on the Illumina HiSeq 4000. Base‐calling of all sequence data was performed using Illumina's RTA v2.7.7.

For the first transcriptome experiment, preparation of TruSeq libraries, subsequent clean-up, quality control and quantification were performed as described in [54 ]. Two pools of 15 libraries were each loaded onto a lane of an Illumina HiSeq 4000 flow cell and sequenced in a 1 × 50 bp single read format. Base calling, demultiplexing, and conversion to FastQ format was as described in [54 ]. For the second experiment, QuantSeq 3′-mRNA FWD libraries (Lexogen) were prepared by the Cornell University, Institute of Biotechnology, Genomics Facility using the manufacturers guidelines. Quality control and quantification of completed libraries was performed using a combination of Qubit dsDNA HS and Advanced Analytical Fragment Analyzer High Sensitivity DNA assays. The libraries where then loaded on a single Illumina NextSeq500 lane and sequenced in a 1 × 86 bp single end format. Base calling was achieved by Illumina RTA v2.4.11 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.18.

Cells were harvested at 80% confluency, 24 hr after a media change. Total RNA was extracted using the Qiagen RNeasy Kit. RNA quality was confirmed using a Fragment Analyzer^TM (Advanced Analytical Technologies). cDNA library preparation was performed using the NEBNext Ultra directional RNA library Prep Kit (Illumina). DNA sequencing was conducted on the Illumina NextSeq 500 system. Image analysis, base calling and quality checks were undertaken with the Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.17. Read data was mapped to the reference sequence Homo_sapiens.GRCh37.75 using a short read aligner after trimming for adapter sequences with Trimmomatic v0.30. A default mismatch rate of 2% was used. Read counts per gene were imported into R Statistical Package v3.2.3 (www.r-project.org), analyzed using LIMMA package v3.26.9 and voom transformed. Genes differentially expressed between cell lines were identified using linear regression models. p-values were adjusted using the Benjamini and Hochberg's method. GO process enrichment was computed using MetaCore^TM v6.27 for all differentially expressing genes with a FDR p-value < 0.05.

RNA sequencing was performed by Service XS (GenomeScan) using the Illumina Next Generation Sequencing Technology. The NEBNext® Ultra II DNA Library Prep kit for Illumina (cat# NEB #E7645S/L) was used to process the samples. Fragmentation of the DNA using the Biorupor Pico (Diagenode), ligation of sequencing adapters, and PCR amplification of the resulting product was performed according to the procedure described in the NEBNext Ultra DNA Library Prep kit for Illumina Instruction Manual. The quality and yield after sample preparation was measured with the Fragment Analyzer. The size of the resulting product was consistent with the expected size of approximately 500–700 base pairs. Clustering and DNA sequencing using the Illumina NextSeq500 was performed according to manufacturer’s protocols. A concentration of 1.6 pM of library was used. NextSeq control software 2.2.0 was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA v2.4.11 and Bcl2fastq v2.20.