Agilent bioanalyzer dna 1000 chip

cDNA library for RNA-Seq was generated from 1 μg total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, poly-A tailed RNAs were purified by oligodT conjugated magnetic beads and fragmented on 94 C degree for 8 minutes, then 1^st strand cDNA was transcribed using random primers and SuperScript II reverse transcriptase (Lifetechnologies, Carslbad, CA, USA). Following this step second strand cDNA synthesized, double stranded cDNA end repaired and 3′ ends adenylated then Illumina index adapters were ligated. After adaptor ligation enrichment PCR was performed to amplify adaptor ligated cDNA fragments. Fragment size distribution and molarity of libraries were checked on Agilent BioAnalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA). Paired read 100bp sequencing runs were performed on Illumina HiScan SQ instrument (Illumina, San Diego, CA, USA).

Paired-end libraries were prepared using NEBNext DNA sample preparation kit following the manufacturer's protocol (New England Biolab). Briefly, DNA was fragmented using the Covaris E210 sonicator to generate double-stranded DNA fragments with a fragment size of 400–500 bp. The ends were repaired, phosphorylated, followed by 3′ end adenylation. Paired end DNA adaptors were ligated and the resulting constructs size selected for ∼500 bp fragments. The excised gel band was purified following manufacturer's protocol utilizing Qiagen Gel Extraction Kits. These fragments were enriched with 12 cycles of PCR. The concentration and size distribution of the libraries was determined on an Agilent Bioanalyzer DNA 1000 chip.
Libraries were loaded onto paired end flow cells and sequenced as 101 by 2 paired end indexed reads on Illumina HiSeq 2000 and base-calling performed using Illumina's RTA version 1.7.45.0.

cDNA library for RNA-Seq was generated from 1 μg total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, poly-A tailed RNAs were purified by oligodT conjugated magnetic beads and fragmented on 94 °C for 8 min, then 1st strand cDNA was transcribed using random primers and SuperScript II reverse transcriptase (Lifetechnologies, Carslbad, CA, USA). Following this step second strand cDNA were synthesized, then double stranded cDNA molecules were end repaired and 3′ ends adenylated then Illumina index adapters were ligated. After adapter ligation step enrichment PCR was performed to amplify the adapter-ligated cDNA fragments. Fragment size distribution and molarity of libraries were checked on Agilent BioAnalyzer DNA1000 chip (Agilent Technologies, Santa Clara, CA, USA). 10pM of denatured libraries were used for cluster generation on cBot instrument, and then sequencing run was performed on Illumina HiSeq 2000 instrument (100 bp paired-end) (Illumina, San Diego, CA, USA).

To prepare samples for small-RNA sequencing, total RNA was extracted using an AllPrep nucleic acid extraction kit (Qiagen, Valencia, CA). RNA was quantified using the RiboGreen fluorometric assay (Thermo Fisher, Waltham, WA). RNA integrity was then measured using a model 2100 Bioanalyzer (Agilent, Santa Clara, CA). Library creation and sequencing were performed by the Mayo Clinic Genome Analysis Core. Briefly, small-RNA libraries were prepared using 1 µg of total RNA per the manufacturer’s instructions for the NEBNext multiplex small-RNA kit (New England Biolabs, Ipswich, MA). After purification of the amplified cDNA constructs, the concentration and size distribution of the PCR products were determined using an Agilent (Santa Clara, CA) Bioanalyzer DNA 1000 chip and Qubit fluorometry (Invitrogen, Carlsbad, CA). Four of the cDNA constructs are pooled, and the 120- to 160-bp miRNA library fraction is selected using Pippin Prep (Sage Science, Beverly, MA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip and Qubit fluorometry. Sequencing was performed across 4 lanes on an Illumina HiSeq 2000 instrument (paired end).

RNA-seq experiments were performed in biological duplicates following the procedures as below. Three milliliters of cells from mid-log phase cultures were mixed with 6 mL RNAprotect Bacteria Reagent (Qiagen). Samples were mixed immediately by vortexing for 5 s, incubated for 5 min at room temperature, and then centrifuged at 5000 g for 10 min. The supernatant was decanted, and any residual supernatant was removed by inverting the tube once onto a paper towel. Total RNA samples were then isolated using RNeasy Plus Mini kit (Qiagen) following the manufacturer's instructions. Samples were then quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and quality of the isolated RNA was checked by running RNA 6000 Pico Kit using Agilent 2100 Bioanalyzer (Agilent). Paired-end, strand-specific RNA-seq library was prepared using KAPA RNA Hyper Prep kit (KAPA Biosystems), following the instruction (24 (link),25 (link)). Resulting libraries were analyzed on an Agilent Bioanalyzer DNA 1000 chip (Agilent). Sequencing was performed on a Hiseq 2500 (Illumina) following the manufacturer's instructions.

RNA-seq was performed using two biological replicates (Dataset S5). The strains were grown under the same conditions as those used in the ChIP-exo experiments. Transcripts were stabilized by mixing 3 ml of cell cultures at the mid-log phase with 6 ml of RNAprotect Bacteria Reagent (Qiagen). Samples were immediately vortexed for 5 s, incubated for 5 min at room temperature, and then centrifuged at 5000 × g for 10 min. The supernatant was decanted, and any residual supernatant was removed by inverting the tube once onto a paper towel. Total RNA samples were then isolated using a RNeasy Plus Mini kit (Qiagen) following the manufacturer's instruction. Samples were then quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific) and quality of the isolated RNA was checked by running RNA 6000 Pico Kit using an Agilent 2100 Bioanalyzer (Agilent). Paired-end, strand-specific RNA-seq libraries were prepared using KAPA RNA Hyper Prep kit (KAPA Biosystems), following the instructions (20 (link),21 (link)). Resulting libraries were analyzed on an Agilent Bioanalyzer DNA 1000 chip (Agilent). Sequencing was performed on a Hiseq 2500 sequencer (illumina) at the Genomics Core facility of University of California, San Diego.