Comment[ArrayExpressAccession] E-MTAB-1901 MAGE-TAB Version 1.1 Investigation Title 14028s araC RNA-Seq Comment[Submitted Name] 14028s araC RNA-Seq Experiment Description We used RNA-seq to determine the effects of AraC and arabinose on RNA levels genome-wide in S. enterica. Wild-type or delta araC mutant cells were grown in both the presence and absence of 0.2% L-arabinose. RNA-Seq libraries from two independent biological replicate samples were sequenced for (i) wild-type cells with no treatment, (ii) wild-type cells treated with arabinose, (iii) delta araC cells with no treatment, (iv) delta araC cells treated with arabinose. Comparisons were made between (i) and (ii) [analysis file name: Salmonella RNA-seq processed WT without arabinose vs WT with arabinose.xls], between (i) and (iii) [analysis file name: Salmonella RNA-Seq proessed no arabinose WT vs no arabinose delta araC.xls], and between (ii) and (iv) [analysis file name: Salmonella RNA-Seq processed arabinose WT vs arabinose delta araC.xls]. All analysis files are available from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1901/E-MTAB-1901.additional.1.zip Experimental Design strain_or_line_design in_vivo_design co-expression_design Comment[AEExperimentType] RNA-seq of coding RNA Comment[AEExperimentDisplayName] RNA-seq of coding RNA from salmonella enterica typhimurium wild-type or delta araC mutant cells grown in the presence or absence of 0.2% L-arabinose to determine the effects of AraC and arabinose on RNA levels genome-wide Experimental Factor Name genotype compound dose Experimental Factor Type genotype compound dose Quality Control Type biological replicate Quality Control Term Source REF EFO Public Release Date 2013-10-04 Person Last Name Wade Person First Name Joseph Person Mid Initials T Person Email jwade@wadsworth.org Person Phone 518-474-2727 Person Address CMS 5227, 120 New Scotland Ave, Albany, NY 12208 Person Affiliation Wadsworth Center, NY DOH Person Roles submitter PubMed ID 24272778 Publication Author List Anne M. Stringer, Salvatore Currenti, Richard P. Bonocora, Catherine Baranowski, Brianna L. Petrone, Michael J. Palumbo, Andrew E. Reilly, Zhen Zhang, Ivan Erill, Joseph T. Wade. Publication Title Genome-Scale Analyses of Escherichia coli and Salmonella enterica AraC Reveal Noncanonical Targets and an Expanded Core Regulon Publication Status under review Protocol Name P-MTAB-35008 P-MTAB-35009 P-MTAB-35010 P-MTAB-35011 P-MTAB-35012 P-MTAB-35013 P-MTAB-35014 Protocol Type growth protocol treatment protocol nucleic acid extraction protocol nucleic acid library construction protocol nucleic acid sequencing protocol high throughput sequence alignment protocol normalization data transformation protocol Protocol Description Overnight cultures were grown in LB 37 degreesC/225rpm and then subcultured 1:100. 1 ml cells were grown in LB +/-0.2% arabinose at 37 degrees C to an OD600 of 0.6-0.8. Duplicate samples were prepared from independent biological replicates for each condition/strain. grown in LB + 0.2% arabinose RNA was purified from cells using a modified version of the hot phenol method that has been described previously. 1 ml cells were mixed with 400 _l ice-cold 95% ethanol and 5% phenol/chloroform/isoamyl alcohol (25:24:21 mix). Cells were pelleted in a microcentrifuge for 1 minute at full speed and washed once with TBS. Cell pellets were resuspended in 400 _l RNA lysis buffer (2% SDS, 4 mM EDTA) and boiled for 3 minutes. 400 uL acid phenol/choroform/isoamyl alcohol mix (pH 4.3) was added and incubated at 65 degrees C for 6 minutes and on ice for 5 minutes. Samples were centrifuged and the aqueous layer was extracted once more with phenol/choroform/isoamyl alcohol mix (pH 4.3). RNA was precipitated with 1 ml 100% ethanol and 40 _l 3 M sodium acetate. RNA was pelleted in a microcentrifuge for 10 minutes at full speed and washed once with room-temperature 75% ethanol. RNA pellets were air-dried and resuspended in water. DNase treatment and Ribosomal RNA removal: A 50uL reaction including 8-10ug RNA, 5uL of 10X buffer and 1uL DNase enzyme (Ambion) was incubated 30 min at 37 degreesC. The reaction was stopped by adding 352uL DEPC H2O + 134uL acid phenol:chloroform + 268uL chloroform:isoamyl alcohol 24:1. After spinning in a microcentrifuge at max spped for 6 minutes, the upper phase was transferred to new microcentrifuge tube containing 40uL 3M Na Acetate + 2uL glycogen (20mg/mL stock). 1mL of ice-cold EtOH was added and the RNA was precipitated at -20 degreesC overnight or 15min at -80 degreesC. The samples were microcentrifuged for 30 min at 4 degreesC at max speed and then washed twice with 0.7mL of 70% EtOH, microcentrifuging 5 min at max speed at 4 degreesC between each wash. The pellets were dried at room temperature and resuspended in 15uL nuclease free water. The ribosomal RNA was removed using the RiboZero kit from EpiCentre. The magnetic beads were washed 2x in RNase free water, and then resuspend in Resuspension Solution. 32.5uL beads and 0.5uL RNase inhibitor were added to each tube. In final volume of 20uL, the following mixture was prepared: 2.5ug DNase treated RNA + 2uL Ribozero reaction buffer + 4uL Ribozero rRNA removal solution. The mixture was incubated at 68 degreesC for 10 min followed by 20C for 5 min. The RNA was then added to the prepared beads and mixed by pipetting and vortexing for 10 s. The samples were incubated at RT for 5 min. The samples were then vortexed for 5 s then incubated at 50 degreesC for 5 min. The tubes containing magnetic bead/RNA mixture were placed on a magnetic stand for 1 min. The RNA (depleted of ribosomal RNA) was transferred to a new tube. The volume was brought up to 180uL with DEPC H2O. 18uL 3M Na Acetate + 2uL glycogen (20mg/mL stock) were added to each RNA sample. 0.6mL ice cold EtOH was added to each sample and the RNA was precipitated at -20 degreesC overnight. The samples were microcentrifuged for 30 min at 4 degreesC at max speed and then washed twice with 0.7mL of 70% EtOH, microcentrifuging 5 min at max speed at 4 degreesC between each wash. The pellets were dried at room temperature and resuspended in 8uL nuclease free water. Prepare libraries using EpiCentre ScriptSeq v2 kit following kit protocol. Fragment RNA and anneal cDNA synthesis primer in 12uL reaction containing nuclease free water + 40ng rRNA depleted RNA + 1uL RNA Fragmentation solution + 2uL cDNA Synthesis primer. Incubate at 85 degreesC for 5 min then place tubes on ice. Synthesize DNA by adding 3uL cDNA Synthesis Premix + 0.5uL 100mM DTT + 0.5uL StarScript Reverse Transcriptase. Incubate at 25 degreesC for 5 min then 42 degreesC for 20 min. Cool tubes to 37 degreesC. Add 1uL Finishing solution and incubate at 37 degreesC for 10 min then 95 degreesC for 3 min. Cool tubes to 25 degreesC then add 7.5uL Terminal Tagging Premix + 0.5uL DNA Polymerase to terminally tag the cDNA. Incubate at 25 degreesC for 15 min, 95 degreesC for 3 min, then cool to 4 degreesC. Purify the cDNA using the Qiagen MinElute PCR Purification Kit - Add 5V Buffer PB to sample and transfer all to MinElute column. Spin 1 min at max speed. Discard flow through. Add 0.75mL Buffer PE to column and spin 1 min at max speed. Discard flow through. Spin column for 1 min at max speed then transfer column to new tube. Add 23.4uL Buffer EB to column, incubate 1 min at room temp, then spin 1 min at max speed. Amplify library by setting up 50uL PCR using 22.5uL purified DNA. Add 25uL FailSafe PCR Premix E + 1uL Forward PCR primer + 1uL ScriptSeq Index PCR Primer + 0.5uL FailSafe PCR Enzyme (1.25U). Heat at 95 degreesC for 1 min, then 12 cycles of 95 degreesC for 30 sec, 55 degreesC for 30 sec and 68 degreesC for 3 min. Incubate at 68 degreesC for 7 minutes. Purify sample using AMPure XP Purification protocol. Add 50uL beads to each pcr sample. Mix by pipetting 10x. Transfer beads+sample to new tube and incubate at room temp for 15 min. Put tubes on magnetic stand for 5 min then remove supernatant. Wash beads 2x, each time with 0.2mL of 80% EtOH, let stand 1 min then remove supernatant. Air dry beads at room temp for 15 min. Add 12uL nuclease free water, remove tubes from magnetic stand and resuspend beads in water. Incubate at room temp for 2 min. Place tubes on magnetic stand for 5 min. Library is in supernatant. Transfer to new tube. Quantify using Qubit. HiSeq 51 nucleotide single end read, using a HiSeq2000 instrument. Bowtie 2.02, default settings Two independent biological replicate samples were analyzed for (i) wild-type cells with no treatment, (ii) wild-type cells treated with arabinose, (iii) delta araC cells with no treatment, (iv) delta araC cells treated with arabinose. Comparisons were made using Cufflinks 2.02 and CuffDiff (default settings) between (i) and (ii) [Filename: Salmonella RNA-seq processed WT without arabinose vs WT with arabinose.xls], between (i) and (iii) [Filename: Salmonella RNA-Seq proessed no arabinose WT vs no arabinose delta araC.xls], and between (ii) and (iv) [Filename: Salmonella RNA-Seq processed arabinose WT vs arabinose delta araC.xls]. Column 1: test_id = Cufflinks ID; Column 2: gene_id = Cufflinks ID; Column 3: PATRIC gene name = systematic gene name assigned by PATRIC ; Column 4: locus = reference genome and corresponding coordinates; Column 5: RefSeq gene name = where possible, a RefSeq gene name has been matched to the PATRIC gene name; Column 6: Commone name = where possible, a common gene name has been matched to the PATRIC gene name; Column 7: sample_1 = description of first group of samples being compared; Column 8: sample_2 = description of second group of samples being compared; Column 9: status = flags genes with high data, low data, etc.; Column 10: value_1 = Cufflinks score for sample_1; Column 11: value_2 = Cufflinks score for sample_2; Column 12: log2(fold change) = difference between value_1 and value_2 (log, base 2); Column 13: test_stat = value used for statistical analysis; Column 14: p_value; Column 15: q_value = false discovery rate; Column 16: significant = yes/no indication of whether the q_value <0.05;
All analysis files are available from https://www.ebi.ac.uk/arrayexpress/files/E-MTAB-1901/E-MTAB-1901.additional.1.zip Protocol Term Source REF EFO EFO EFO EFO EFO EFO EFO Protocol Hardware Illumina HiSeq 2000 Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo http://www.ebi.ac.uk/arrayexpress Comment[AdditionalFile:xls] Salmonella_RNA-Seq_processed_arabinose_WT_vs_arabinose_delta_araC.xls Salmonella_RNA-seq_processed_WT_without_arabinose_vs_WT_with_arabinose.xls Salmonella_RNA-Seq_proessed_no_arabinose_WT_vs_no_arabinose_delta_araC_.xls Comment[SecondaryAccession] ERP003981 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR348243-ERR348250 SDRF File E-MTAB-1901.sdrf.txt