title: GSM4435421: beta'WT_RNET-seq_rep1; Escherichia coli str. K-12 substr. W3110; OTHER
library_construction_protocol: The cells collected were resuspended and lysed by 120 kU Ready-Lyse lysozyme (Lucigen), 400 U RNase I (Invitrogen) and 40 U alkaline phosphatase (NEB) at room temperature for 10 min. The chromosomal DNA with RNAP binding was pelleted and treated with 300 U RNase I, 6 U Turbo DNase (Invitrogen) and 100 U DNase I (Roche) by vortexing at room temperature for 10 min. After centrifugation, the supernatant containing ECs was transferred to mix with Ni-NTA beads for 1 hour at 4 ºC with continuous shaking (1000 rpm). The beads were washed and ternary complexes immobilized on the beads were further digested by 100 U RNase I, 2 U Turbo DNase and 40 U DNase I for 30 min at room temperature. The beads were washed and loaded onto 0.5 ml Ultrafree-MC centrifugal filters (Millipore). The immobilized ECs were eluted with wash buffer containing 0.3 M imidazole. The nucleic acids in the eluates were extracted once with phenol:chloroform:isoamylalcohol (PCI; 25:24:1) and once with chloroform. The top water phase was collected and the nucleic acids were precipitated by isopropanol at -80 ºC for 30 min. After centrifugation, the nucleic acids pellet was washed by 80% ethanol and air dried. The resultant pellet was dissolved in nuclease-free water. The DNA was removed by 2 U Turbo DNase and 10 U DNase I at 37 ºC for 15 min. The residual RNA was extracted by PCI and precipitated by isopropanol. Libraries were constructed based on the modified protocol of NET-seq (Mayer, A. and Churchman, L. S. Nat Protoc. 2016). Briefly, a barcode DNA linker was ligated to 3' end of nascent RNA. The resulting RNA-DNA chimera was reverse transcribed by using PrimeScript Reverse Transcriptase and a 5' phosphorylated reverse transcription primer. The cDNA between 75-100 nt were excised after running 10% Urea-TBE gel, extracted and precipitated by isopropanol. The purified cDNA was circularized by ssDNA ligase (Takara) and applied to PCR the sequencing library using Illumina index primers and PrimeSTAR Max DNA polymerase (Takara). The DNA product excised from gel was extracted and purified for Illumina sequencing.
attributes.tag: GEO Accession
attributes.value: GSM4435421