title: GSM5695801: spike-in_pool4; synthetic construct; RNA-Seq
library_construction_protocol: Samples have been either extracted by RNAsnap (Stead et al., NAR 2012), Hot Phenol, RiboPure (ThermoFisher/Ambion), RNeasy (Qiagen) or Trizol, precipitated, Dnase treated and cleaned uo with RNA clean & concentrator columns (Zymo research). RNA-seq for E. coli Δpnp, Δrnb, ΔpcnB and MG1655 wildtype was executed as described in (Parker et al., Cell Systems 2020). 5' end-sequencing of 5' phosphorylated RNAs was done modifying the RNA-seq protocol slightly. Namely, prior RNA fragmentation a 5' end an RNA oligo (ACGACGCUCUUCCGAUCUNNNNNNNN) was ligated to the RNA 5' end and precipitated. RNA was fragmented and size selected in the size range of 41-71nt. 3' end dephosphorylation and adenylation to enable uniform reverse transcription, reverse transcription, cDNA size selection again and PCR were done as described in (Parker et al., Cell Systems 2020). Rend-seq libraries were prepared as described in (Lalanne et al., Cell 2018) with 25s RNA fragmentation. For 3' end sequencing the Rend-seq protocol was modified in a way that 3' end ligation (5'_App/CTGTAGGCACCATCAAT/3ddC_3') was done preceding fragmentation for 2min. Final libraries were sequenced on Illumina HiSeq 2000 or NextSeq500 machines.