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Sample GSM7310631

Query DataSets for GSM7310631

Status

Public on May 26, 2023

Title

Tre3

Sample type

SRA

Source name

R20296

Organism

Clostridioides difficile R20291

Characteristics

strain: R20291
growth phase: mid-logarithmic phase
treatment: Trehalose grown

Growth protocol

BHIS overnight R20291 cultures of each were sub-cultured to an OD600 of 0.05 in 25 mL of DMM supplemented with either 20 mM glucose, 10 mM trehalose or 20 mM glucose and 10 mM trehalose. These were grown to mid-logarithmic phase (strain dependent 4-6 h), mixed with an equal volume of ice-cold 1:1 ethanol: acetone mix and stored at -80oC.

Extracted molecule

total RNA

Extraction protocol

The cell suspension was thawed on ice, centrifuged at 5 000 x g for 10 min at 4oC, supernatant decanted and the pellet resuspended in 5 mL 1% beta-mercaptoethanol. The suspension was then centrifuged once more at 10 000 x g for 5 min at 4oC. RNA isolation was carried out using a Qiagen RNeasy Mini Kit (Qiagen, Manchester, UK ). Cells were resuspended in 200 µL TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0) containing 15 mg/mL lysozyme and 1.5 mg/ml Proteinase K (Qiagen, Manchester, UK) and incubated for 10 min at room temperature followed by the addition of 700 µL buffer RLT with 1% beta-mercaptoethanol. Cells were then transferred to a 2 mL Lysing Matrix B tube (MP Biomedicals, Cambridge, UK) and treated in a FastPrep-24 instrument (MP Biomedicals, Cambridge, UK) at a setting of 6.0 m/s for 45 s. The lysate was then centrifuged at 13 000 x g for 10 min and the supernatant added to 500 µL of 98% ethanol before RNA isolation and clean-up using a RNeasy column by following the manufacturer’s instructions. The protocol was modified to include an initial on-column DNA digestion step using the RNase-free DNase set (Qiagen, Manchester, UK) according to the manufacturer’s instructions.
RNA integrity was assessed using the Agilent 4200 TapeStation and the Agilent RNA ScreenTape Assay (Agilent, SantaClara, USA) with RNA samples of RIN > 8.0 being used for subsequent procedures. Ribodepletion was performed on 500 ng of total RNA, using the Qiagen FastSelect-5S/16S/23S ribodepletion kit (Qiagen, Manchester, UK) using the protocol: FastSelect-5S/16S/23S with NEBNext Ultra II Directional Library Prep Kit. Indexed sequencing libraries were then prepared using the NEBNext Ultra II Directional RNA Library Preparation Kit for Illumina (New England BioLabs, Ipswich, UK) and NEBNextMultiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (New England Bio Labs, Ipswich, UK). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (Thermo Fisher Scientific, Massachusetts, USA). Library fragment-length distributions were analysed using the Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent, Santa Clara, USA). Libraries were pooled in equimolar amounts and final library quantification performed using the KAPA Library Quantification Kit for Illumina (Roche; Diagnostics, UK). The library pool was sequenced on the Illumina NextSeq 500 on a NextSeq500 High Output 300 cycle kit (Illumina, San Diego, USA), to generate over 10 million pairs of 150-bp paired-end reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Differential gene expression analysis was carried out by mapping the sequenced reads to the C. difficile R20291 reference genome (https://www.ncbi.nlm.nih.gov/nuccore/FN545816.1). Reads were filtered by using the trimming pipeline, which filtered low sequencing score and reads aligned to adapter sequences. Raw reads were trimmed against adaptors and then reads were quality trimmed by skewer. Reads that passed the filter were then mapped onto the reference genome by the hisat2 mapping tool. Mapped reads were then counted by using the “featurecounts” tool (http://subread.sourceforge.net/) to determine the number of uniquely and correctly aligned reads per gene. Normalised read counts (RPKM) for a given gene were determined by normalising the exon space of a gene against the total number of mapped reads (excluding rRNA and reads not uniquely mapped according to their mapping quality score [MAPQ] using MAPQ20) in an alignment file and against the total length of the gene’s exon space. Differentially expressed genes were then determined using the DESeq package, which analyses the variance between biological replicates within the RNA-seq analysis in order to better model the expression values of individual genes within the group of replicates. DESeq then determines differentially expressed genes for each comparison by using, p value= ≤ 0.05.
Assembly: FN545816.1
Supplementary files format and content: Excel file shows transcripts per million (TPM) which is equivalent to RPKM

Submission date

May 09, 2023

Last update date

May 26, 2023

Contact name

Andrew Marshall

Organization name

Queen's University Belfast

Street address

19 Chlorine Gardens

City

Belfast