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Sample GSM4037721

Query DataSets for GSM4037721

Status

Public on Feb 21, 2020

Title

RNAseq_C_lysate2

Sample type

SRA

Source name

cell cultures

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

rna purified from: whole cell lysate
growth conditions: 37 C OD600 = 0.5

Growth protocol

Unless specified, cells were cultured at 37 °C in 500 mL of LB + ampicillin (50 mg/L). IPTG was added (0.3 mM final concentration) when the culture reached OD600 = 0.3 and cells were harvested by filtration at OD600 = 0.5. To induce cold shock, cells were cultured at 37 °C in 175 mL of LB + ampicillin to OD600 = 0.65, induced with IPTG for 20 min at 37 °C, then mixed with 325 mL of ice-cold LB + ampicillin (50 mg/L) + IPTG (0.3 mM) and cultured at 10 °C for 30 min. To prepare samples in stationary phase, cells were cultured at 37 °C in 500 mL of LB + ampicillin to OD600 = 2.0, then further cultured at 37 °C for 4 h. IPTG was added to the culture and cells were further incubated for 1 h. For profiling with retapamulin, cells were grown at 37 °C in 500 mL of LB + ampicillin to OD600 = 0.3, induced with IPTG, grown to OD600 = 0.45, and then harvested by filtration 5 min after the addition of retapamulin (100 µg/mL final concentration).

Extracted molecule

total RNA

Extraction protocol

Cell pellets were cryogenically pulverized in a Spex 6870 freezer mill; 5 cycles of 1 min at 5 Hz with 1 min cooling. Cell lysates were clarified by centrifugation. For standard ribosome profiling (StRP), lysates were digested with nucleases, and monosomes were purified over a sucrose gradient. For MS2 ribosome profiling (MS2RP), monosomes were purified by pulldown with MS2 coat protein.
For StRP and MS2RP, ribosome footprints were PAGE purified, ligated to a linker using RNA ligase T2, converted to DNA using RT, circularized, and PCR amplified. For RNAseq, libraries were constructed by TruSeq Stranded Total RNA Gold

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

ks65
mRNA
RNAseq_C_lysate2_minus.wig
RNAseq_C_lysate2_plus.wig

Data processing

Perfectly matching reads (including 5’-end and 3’-end UMI) were converted to a single read. The linker sequence was trimmed and reads mapping to rRNA, tRNA, ssrA, ssrS, lacI, or ffs sequences were discarded. The remaining reads were mapped to the E. coli K-12 MG1655 genome.
Genome_build: NC_000913.2
Supplementary_files_format_and_content: WIG files of ribosome occupancy, assigned to the 3'-end of reads, normalized by the total number of reads in the library.

Submission date

Aug 16, 2019

Last update date

Feb 21, 2020

Contact name

Allen R Buskirk

E-mail(s)

buskirk@jhmi.edu

Organization name

Johns Hopkins University School of Medicine

Department

Molecular Biology and Genetics