GSM3785159: ChIP-seq stationary phase input for rep2; Escherichia coli; ChIP-Seq
For the RNA-seq samples, the E. coli cells were quenched by adding 0.5 volume of cold Stop Buffer (5% phenol in ethanol) immediately to the culture medium before harvest and stay on the ice for 15 minutes. Cell pellets were collected by centrifugation (6,000 rpm, 5 minutes at 4 °C), thoroughly resuspended in 100 μl of lysozyme solution [2 mg/ml in TE buffer (10 mM Tris-HCl and 1 mM EDTA)], and incubated for 2 minutes. The cells were immediately lysed by adding 1 ml of TRIzol Reagent (Invitrogen, 15596) and the RNA was extracted following manufacture's instruction. For the ChIP-seq samples, the the E. coli cells were fixed with 1% formaldehyde with continued shaking at 37 °C for 10 minutes before quenching with glycine (100 mM final concentration). Cells were then lysed and DNA was sheared by sonication followed by the treatment with micrococcal nuclease and RNase A. Antibody against RNAP β was used for target DNA enrichment. A new RNA-seq method SEnd-seq was developed and used to prepare the SEnd-seq library, standard RNA-seq library was prepared with the TruSeq Stranded mRNA Library Prep Kit (Illumina, RS-122-2101) following the manufacturer's instructions, ChIP-seq library was prepared with NEBNext Ultra II DNA Library Prep Kit (New England BioLabs, E7645)after ChIP DNA enrichment. For SEnd_seq, the RNA was ligated to a RNA adaptor (/5Phos/rNrNrN rNrArArCrCrUrGrCrUrArUrCrArArCrUrG/3ddC/ or /5Phos/rNrNrN rNrGrCrUrUrCrCrUrGrCrUrArUrCrArArCrUrG/3ddC/). The ligated RNA was converted to cDNA with the same Biotinylated RT primer(5GGGCAG T/iBiodT/GATAGCAGG). For RNA-seq , rRNA was depleted with the Ribo-Zero rRNA Removal Kit. The sequencing library was prepared with the TruSeq Stranded mRNA Library Prep Kit following the manufacturer's instructions. For ChIP-seq, the enriched ChIP DNA and input DNA sequencing libraries were prepared with NEBNext Ultra II DNA Library Prep Kit.
GEO Accession
GSM3785159