GSM9008959: MG1655 cells, 37˚C, 6 min rifampicin treated, total RNA-seq; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
Total RNA was purified from samples using hot acid phenol extraction. Briefly, frozen pellets were resuspended in 500 µL of RNA extraction buffer (0.3M NaOAc, 1mM EDTA, 1% SDS) and mixed with 600 µL hot phenol (Invitrogen, AM9720) at 65°C for 5 min. The mixture was centrifuged at 14000 rpm at 4°C for 10 min, and the upper aqueous layer was transferred to a new tube. A second phenol/chloroform extraction was performed, followed by an additional extraction with an equal volume of chloroform. The upper layer was mixed with 2 µL GlycoBlue coprecipitant (Invitrogen, AM9516) and an equal volume of isopropanol to precipitate RNA. RNA pellets were washed with 750 µL 80% ethanol, dried, and resuspend in 10 mM Tris pH 7. Genomic DNA was removed by Turbo DNase (Invitrogen, AM2238). Purified RNA was fragmented by mixing with an equal volume of 2x alkaline fragmentation buffer (0.6 volumes of 100 mM Na2CO3 and 4.4 volumes of 100 mM NaHCO3), incubated at 95°C for 13 min, and then purified using Zymo oligo CC kit (Zymo Research, D4060). 600 ng fragmented RNA was dephosphorylated by T4 polynucleotide kinase (New England Biolabs, M0201) at 37°C for 1 hour, followed by ligation to 3' linker oligos (sequences provided in the publication) using T4 RNA ligase 2 truncated KQ (New England Biolabs, M0373) at 16°C overnight. The ligated products from different samples were pooled and purified by size excision. For samples requiring mRNA enrichment, ribosomal RNAs were removed with Ribo-Zero rRNA depletion kit (Epicentre, MRZGN126). Ligated RNA was reverse transcribed into cDNA by SuperScript IV Reverse Transcriptase (Invitrogen, 18090010). The resulting cDNA was purified and ligated to a second oligo (sequences provided in the publication) using T4 RNA ligase 1 (New England Biolabs, M0204) at 16°C overnight. The final ligation products were purified by size excision. The purified cDNAs were PCR amplified using Phusion polymerase (New England Biolabs, M0530) (PCR primers provided in the publication). The final libraries were size selected and purified. Sequencing was performed on an Illumina HiSeq 4000 system, using single-end 50-nucleotide reads.