alias: GSM3681040
title: GSM3681040: WT_15; Escherichia coli K-12; RNA-Seq
library_construction_protocol: RNA was extracted by hot trizol lysis and Direct-zol RNA MiniPrep (Zymo). rRNAs were removed using the Ribo-Zero Kit for Gram-negative bacteria (Illumina) and mRNAs were then fragmented with RNA fragmentation reagents (Ambion). First-strand cDNA synthesis was performed with random primers (Invitrogen) and Superscript III. After purifying cDNAs by phenol:chloroform:isoamyl alcohol extraction and precipitation, second-strand synthesis occurred with dUTP (Promega) instead of dTTP and RNase H (NEB), E.coli DNA ligase (NEB), and DNA Pol I (NEB). Sequencing libraries were built as with other libraries, except that the second strand was digested with USER (NEB) after the addition of Y-adaptors. Libraries were constructed by end repairing DNA with T4 DNA polymerase (NEB), T4 PNK (NEB), and Klenow large fragment (NEB). 3' overhangs were added with Klenow (3'→5' exo-) (NEB). Y-shaped Illumina adapters were ligated onto the DNA, and libraries were amplified with either Phusion (NEB) or Kapa HiFi Polymerase (Kapa Biosystems). Libraries were size selected to be 250-600 bp.
identifiers.namespace: GEO
identifiers.id: GSM3681040