GSM5839618: MB7180 strain (DHB4 ΔdegP + pAL-FevRCit-CYTO-FLAG) treated at 42°C, replicate 3; Escherichia coli K-12; RNA-Seq
1.5ml of each culture was harvested by centrifugation and total RNA was extracted using RNAeasy Mini Kit (Qiagen cat#74104) including the DNAse treatment step with RNase-Free DNase Set (Qiagen cat#79254) following the vendor guidelines. An extra DNase treatment step was added after extraction using DNase I (RNase-free, New England Biolabs cat#M0303S) and RNA was purified using Monarch® RNA Cleanup Kit (New England Biolabs cat#T2040L) eluting in 10µl. Concentration was measured using QubitTM dsDNA BR Assay Kits (Invitrogen cat#Q32850) on a Qubit 2.0 Fluorometer, and the RNA Integrity Number (RIN) was corroborated to be >9 using RNA 6000 Nano Kit (Agilent cat#5067-1511) on an Agilent 2100 Bioanalyzer System. For samples MB7409, MB7410 and MB7434 treated at 30°C: library preparation, sequencing and analysis was performed by CD genomics. Firstly, the ribosomal RNA depletion was performed using NEBNext® rRNA Depletion Kit (Bacteria, New England Biolabs cat#E7860S). The rRNA-depleted RNA was purified by 2x RNAClean XP beads (Beckman Coulter) and eluted in 45ul of nuclease-free water. The purified RNA was then mixed with 4ul of NEBNext First Strand Synthesis Reaction Buffer and 1ul of random primers. The reaction was incubated at 94C for 12 mins for fragmentation. Subsequently, to perform first strand cDNA synthesis, 8ul of NEBNext Strand Specificity Reagent and 2ul of NEBNext First Strand Synthesis Enzyme Mix was added to the reaction and the reaction was incubated at 25C 10 mins, 42C 30 mins, 70C 15 mins. Second strand reaction was then performed by adding 8ul of NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix (10X), 4ul of NEBNext Second Strand Synthesis Enzyme Mix, and 48ul of nuclease-free water. The reaction was incubated at 16C 1hr. The cDNA was purified by 1.8x SPRIselect Beads (Beckman Coulter) and eluted in 50ul of nuclease-free water. The reaction was incubated at 16C 1hr. The cDNA was purified by 1.8x SPRIselect Beads (Beckman Coulter) and eluted in 50ul of nuclease-free water. Subsequently, end prep reaction was performed by adding 7ul of NEBNext Ultra II End Prep Reaction Buffer and 3ul of NEBNext Ultra II End Prep Enzyme Mix into 50 ul purified cDNA. Endprep reaction was incubated at 20C 30 mins and 65C 20 mins. Adaptor ligation reaction was then performed by adding 1ul of NEBNext Ligation Enhancer, 30ul of NEBNext Ultra II Ligation Master Mix, and 2.5ul of NEBNext Adaptor, diluted to 0.5uM in Adaptor Dilution Buffer. The mix was incubated at 20C 15 mins. 3ul of USER Enzymer (New England Biolabs) was then added to the ligation product and the reaction was incubated at 37C 15 mins. The ligated product was purified by SPRIselect Beads (Beckman Coulter) and eluted in 15ul of nuclease-free water. PCR was carried out by adding 25ul of NEBNext Ultra II Q5 Master Mix, 5ul of i5 Primer, and 5ul of i7 Primer into 15ul of purified ligated product. PCR was performed at 98C 30 seconds, 15 cycles of 98C 10 seconds and 65C 75 seconds, and a final extension at 65C 5 mins.  The final library was then purified by SPRIselect Beads and loaded into Illumina NovaSeq6000 paired end 150 bp mode for sequencing. For the rest of the samples: Ribosomal RNA was removed from the total RNA with NEBNext® rRNA Depletion Kit (Bacteria, New England Biolabs cat#E7860S). Libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs cat#E7760L) using oligos for indexing from NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs, New England Biolabs cat#E6440S). Final library quality and concentration was estimated using High Sensitivity DNA Kit (Agilent cat#5067-4626) on an Agilent 2100 Bioanalyzer System and all the libraries were pooled at 4nM in 20uL and sequenced on the Illumina Nextseq 500 Mid Output 2x75+8i+8i  following the manufacturer's protocols.