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Sample GSM3334106

Query DataSets for GSM3334106

Status

Public on Mar 03, 2020

Title

Ribo_L1

Sample type

SRA

Source name

cell culture

Organism

Streptomyces avermitilis

Characteristics

strain: MA-4680
growth phase: late-exponential phase (L)

Treatment protocol

Thiostrepton was treated to cell culture for 5 minutes before harvesting the cells.

Growth protocol

The mycelium of S. avermitilis MA4680 was maintained in 25% glycerol. Cells were first recovered in 50 mL R5- media with 8 g glass beads (3 ± 0.3 mm diameter) at 30 oC, 250 rpm. R5- medium consists of 5.73 g/L TES (pH 7.2), 103 g/L sucrose, 10 g/L glucose, 5 g/L yeast extract, 10.12 g/L MgCl2∙6H2O, 0.25 g/L K2SO4, 0.1 g/L casamino acids, 0.08 mg/L ZnCl2, 0.4 mg/L FeCl3∙6H2O, 0.02 mg/L CuCl2∙2H2O, 0.02 mg/L MnCl2∙4H2O, 0.02 mg/L Na2B4O7∙10H2O, and 0.02 mg/L (NH4)6Mo7O24∙4H2O. The inoculum was then transferred to fresh R5- media with 8 g glass beads for main culture. Cells were harvested at 13, 17, 19.5 and 33.5 hours after inoculation for early exponential phase (E), transition phase (T), late-exponential phase (L) and stationary phase (S), respectively.

Extracted molecule

total RNA

Extraction protocol

The cell pellet was washed with polysome buffer (20 mM Tris-HCl pH 7.4, 140 mM NaCl, 5mM MgCl2, and 33.5 ug/mL thiostrepton) and resuspended with lysis buffer (475 uL Polysome buffer, 25 uL Triton X-100, and 6 uL DNase I (New England Biolabs)). The cell suspension was frozen with liquid nitrogen and lysed by grinding using mortar and pestle. The cell lysate was centrifuged at 4 oC for 10 minutes at 16,000 g and soluble supernatant was recovered. Ribosome unprotected RNA was digested by treating RNase I (Invitrogen) by incubating at 37 oC for 45 minutes. After RNase I digestion, RNase activity was inactivated by treating SUPERase-In (Invitrogen) and monosome was recovered using Sephacryl S-400 column (GE Healthcare Life Science). Ribosome protected RNA was recovered using phenol:chloroform:isoamyl alcohol = 25:24:1 solution and rRNA was removed with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) according to the manufacturer’s instructions. After rRNA depletion, RNA was resolved on a 15% TBE-urea gel and 26-34 nt RNA fragments were size-selected. The size-selected RNA was eluted in 300 mM sodium acetate pH 5.2, 1 mM EDTA and 0.25% SDS. The eluted RNA was further purified with ethanol precipitation.
Library was constructed with NEB Next small RNA library prep set (New England Biolabs) according to the manufacturer’s instructions. The constructed libraries were amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo) for Illumina sequencing. The amplification step was monitored on a CFX96 Real-Time PCR Detection System (Bio-Rad) to be stopped before the PCR reaction was fully saturated. The amplified libraries were further size-selected on 2% agarose gel with MinElute Gel Extraction Kit (Qiagen).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

Sample 19
Ribo_DESeq2.txt

Data processing

Library strategy: Ribo-seq
CLC Genomics workbench 6.5 (CLC bio) was used for data trimming and read mapping.
The adaptor sequence was trimmed from reads of the Ribo-seq libraries before being aligned to the genome. Also, the random 3’-overhanging (N2) sequences in reads of Term-seq library were trimmed.
The reads were aligned to the S. avermitilis genome (BA000030) with the following parameters: mismatch cost 2, deletion cost 3, insertion cost 3, length fraction 0.9, and similarity fraction 0.9.
Only uniquely mapped reads were retained.
The expression of the genes was normalized using the DESeq2 package in R.
The 5' end position of dRNA-seq reads from TAP treated library were considered to be potential TSSs. Briefly, the potential TSSs apart less than 100 bp from the ones located neighboring positions were clustered together. Then, the potential TSSs located adjacent positions in each cluster were sub-clustered together based on the standard deviation of their genomic positions (< 10). Only the potential TSS clusters with more than three read counts were considered and the potential TSSs with maximum read counts within each sub-cluster were selected as TSSs. Then the read counts of selected TSS positions from TAP treated and TAP untreated libraries were compared and positions with more read counts in TAP untreated library were discarded. Then, the selected TSSs were manually inspected using the corresponding RNA-seq profile
The 3' end position of Term-Seq reads located within intergenic regions including up to 10 bp downstream to gene were regarded as potential transcription termination sites (TTS). The positions were clustered together based on the distance from adjacent positions (< 10 bp). Within each cluster, the read count of each position was assumed to follow normal distribution and read count enriched positions were deduced by calculating modified z-score. Z(x)=(r(x)-μ(r(x)))/(σ(x)), where, μ(r(x))=1/(N(x)-1)(-r(x)+∑_(y∈C(x))▒r(y) ), σ(x)=√(μ(〖r(x)〗^2 )-〖μ(r(x))〗^2 ). Z(x) is modified z-score at position x, r(x) is the read count of evaluated position x. μ(r(x)) and σ(x) is the mean and standard deviation of read counts of other positions in the cluster except the evaluated position, respectively. N(x) is the length of the cluster containing position x and C(x) is the set of positions within the cluster containing position x. The positions with read count less than 3 or modified z-score less than 3 were discarded. All the procedures above were conducted separately for each biological replicate. Among the remaining potential TTSs, the reproducible positions with the highest read count within the intersecting region of clusters from two biological replicate was selected as TTS. For example, if genomic positions from 3 to 25 were clustered together for one replicate and genomic positions from 13 to 42 were clustered together for another replicate, potential TTS with highest read count within the genomic positions from 13 to 25 was selected as TTS.
Genome_build: BA000030
Supplementary_files_format_and_content: tab-delimited text files include TSS, TTS or normalized expression values for each sample

Submission date

Aug 15, 2018

Last update date

Mar 28, 2020

Contact name

Yongjae Lee

E-mail(s)

[email protected]

Organization name

KAIST

Street address

291 Daehak-ro