GSM1537646: Escherichia WT Lysate; Escherichia coli; RIP-Seq
The equivalent of 5 OD600 was saved for RNA extraction with TriZOL before adding antibodies (lysate RNA sample). For coIP samples, beads were resuspended in the lysis buffer, mixed with an equal volume of phenol:chloroform:isopropanol (25:24:1, pH4.5, Roth) for 20 s and incubated at room temperature for 3 min. After centrifugation, the aqueous phase was precipitated with isopropanol (coIP RNA sample). The purified RNA coIP sample was treated with DNase I (Thermo Scientific) to remove the residual DNA and reisolated with phenol:chloroform:isopropanol. The spike-in RNA (5’P-CUCGUCCGACGUCACCUAGA, IBA) had been added to 40 pg/µl for coIP samples and to 1.6 ng/µl for lysate samples. RNA-seq libraries were prepared by Vertis AG (Freising-Weihenstephan, Germany). Briefly, RNA was polyadenylated with poly(A) polymerase, 5’-triphosphates were removed with tobacco acid pyrophosphatase followed by ligation of a 5’-adapter. First-strand cDNA synthesis was performed with the use of an oligo(dT) barcoded adapter primer and the M-MVL reverse transcriptase. The resulting cDNA was PCR-amplified with a high fidelity DNA polymerase. cDNA was purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics).
GEO Accession
GSM1537646