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Sample GSM4037747

Query DataSets for GSM4037747

Status

Public on Feb 21, 2020

Title

MS2RPRET_A

Sample type

SRA

Source name

cell cultures

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

rna purified from: MS2 pulldown
growth conditions: 100 ug/mL retapamulin, 5 min

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

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

ks172
mRNA: ribosome footprints
MS2RPRET_A_minus.wig
MS2RPRET_A_plus.wig

Data processing

Library strategy: Ribo-seq
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)

[email protected]

Organization name

Johns Hopkins University School of Medicine

Department

Molecular Biology and Genetics