GSM4564496: CAG67202 pALS14 uninduced [wt_14_un_2]; Escherichia coli BW25113; RNA-Seq
RNA was isolated from frozen cell pellets using Trizol (Invitrogen) extraction according to the manufacturer's protocol. One microgram of purified RNA was fragmented at 95 C for 7 min in 1X T4 RNA Ligase buffer (NEB) with an equal volume of 2X alkaline fragmentation buffer (0.6 volumes of 100 mM Na2CO3 plus 4.4 volumes of 100 mM NaHCO3). After 3'-end healing with PNK (NEB) in T4 RNA ligase buffer for 1 h, 3' ligation to a pre-adenylated, barcoded TruSeq R1 adapter with 5 random bases at its 5' end was performed overnight. The barcoded samples were then pooled and run onto a 6% TBE-Urea gel for size selection (>15 nucleotide insert size), eluted, and ethanol precipitated before performing ribosomal RNA subtraction (RiboZero). Reverse transcription with SuperScript IV (Invitrogen) was performed using a TruSeq R1 RT primer, followed by ligation of the TruSeq R2 adapter to the 3' end of the cDNA overnight, prior to another gel size selection as described above. A final PCR of the library was performed with indexed TruSeq PCR primers to add the index and P5/P7 flowcell adapters, followed by gel extraction, precipitation, and a BioAnalyzer (Agilent) run for quality control before sequencing on a NextSeq500 platform.