 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 11, 2019 |
| Title |
StrainB-TotalmRNA-Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
StrainB-TotalmRNA
|
| Organism |
Mycolicibacterium smegmatis |
| Characteristics |
strain background: M. smegmatis mc2 155
strain: StrainB
genotype/variation: Endogeneous AltRpsR labeled with 3HA tag; Endogeneous CanRpsR labeled with 3Flag tag
molecule subtype: total RNA
|
| Treatment protocol |
Bacterial cultures of both Stain A and Strain B were grown till OD600nm=1 and treated with 100μg/ml chloramphenicol for 3 mins to arreset elongating ribosomes. Bacteria were collected by centrifigation at 4500g, 4°C, 10min.
|
| Growth protocol |
Both Mycobacterium smegmatis Strain A and Strain B were grown in sauton medium supplied with 10μM ZnSO4.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were extracted by miRNeasy kit from bacteria lysate. For ribosome footprint library, bacteria suspension was dropped into liquid nitrogen and smashed into powder. The powder was further pulverized with beads beating 4 to 5 times with chilling in liquid nitrogen between each step. After centrifugation at 18500g for 45 min, the cleared lysate was transferred to a new tube supplied with 70U/ml MNase to digested mRNA not protected my ribosome. The pre-digested lysate was laid over a 1M sucrose cushion and centrifuged at 30,000 rpm, 4°C for 20h in Beckman 70Ti rotor. After ultracentrifugation, the ribosome pellet was washed and dissolved in polysome buffer. The dissolved ribosome solution was further digested with MNase at 4°C for 1h. The digested ribosome fraction was incubated with either Flag resin or HA resin at 4°C overnight. After washing of the bound resin, the resin was elued with 3Flag/3HA peptide to isolate the AltRibo, and the remaing flow-through was confirmed as consisting almost entirly of CanRibo. Ribosome footprints were further extracted by miRNeasy Kit.
For RNA-seq library, rRNA was romoved by Ribo-Zero rRNA Removal Kit and library was constructed by NEBNext Ultra Directional RNA Library Prep Kit for illumina. For ribosome footprint library, footprints were selected for 24-36nt by marker oligo through 15% TBE-UREA PAGE. The corresponding region was sliced and purified. RNA was further dephosphoylated and ligated with linker. The ligation products were purified again by 15% TBE-UERA PAGE. Reverse transcription was followed up as well as circularization. rRNA was depeleted by biotinylated oligo and the rRNA substracted circularized cDNA template was used for library amplification.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
StrainB-totalmRNA-Rep1
processed data file: StrainA-AltRibo_StrainB-AltRibo.gene.TE, StrainA-CanRibo_StrainB-CanRibo.gene.TE, StrainA-TotalRibo_StrainB-TotalRibo.gene.TE
|
| Data processing |
For RNAseq, quality control was performed by FastQC and adaptors were trimmed by FASTX-ToolKit.
For Ribosome profiling, after quality control and adaptor removal, reads that were less than 25nt long were discareded and longer than 36nt were trimmed to 36nt.
Next, Ribosome profiling reads and RNA-seq reads that aligned to rRNAand tRNA were romoved using bowtie2.
The remaining reads were aligned to genome using bowtie2 with "sensitive-local" option.
The mapped reads were normalized to reads per kilobase million (RPKM) using total number of mapped reads.
Genome_build: ASM1500v1
Supplementary_files_format_and_content: Differential translation analysis among different groups and translation effienency of each ribosome fraction normalized by RNA-seq background in different strains. For differential translation analysis, replicates were included. X represents read count of Total Ribo, Y represents read count of experiment set (AltRibo/CanRibo), fold change, p-value were presented in the file. For translation efficiency information, left column represents translation effiency in StrainA, right column represents translation efficiency in strain B.
|
| |
|
| Submission date |
Mar 05, 2019 |
| Last update date |
Apr 11, 2019 |
| Contact name |
Babak Javid |
| E-mail(s) |
bjavid@gmail.com
|
| Phone |
+86-10-62771552
|
| Organization name |
Tsinghua University
|
| Department |
School of medicine
|
| Lab |
Javid Lab
|
| Street address |
Biotechnology Building Room 4302, Tsinghua University, Haidian District
|
| City |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
| |
|
| Platform ID |
GPL26255 |
| Series (1) |
| GSE127827 |
Selective translation by alternative bacterial ribosomes |
|
| Relations |
| BioSample |
SAMN11054007 |
| SRA |
SRX5464667 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
