|
| Status |
Public on May 18, 2016 |
| Title |
C_difficile_Biofilm_3 |
| Sample type |
SRA |
| |
|
| Source name |
Clostridium difficile cells
|
| Organism |
Clostridioides difficile R20291 |
| Characteristics |
growth: biofilm
|
| Growth protocol |
For planktonic samples, overnight culture in brain heart infusion media supplemented with 0.5% yeast extract and 0.1% L-cysteine (BHIS) was mixed 1:10 with fresh BHIS and grown with shaking for 6 h before mixing 1:1 with RNAProtect™ (Qiagen). Biofilm samples were grown for 1 week in glass jars containing 8 mm glass beads. The glass beads were added in order to increase surface area and maximize RNA yields. BHIS medium was changed daily and 6 h prior to cell harvest. To harvest adherent cells (biofilm), media was removed and the beads rinsed with fresh media before covering with RNAProtect™ and subjecting to vigorous shaking for 2 min. For plate growth samples, overnight culture was streaked onto Columbia blood agar plates (BD Biosciences) and kept in the anaerobic chamber for 24 hours prior to a three-day room temperature growth in an anaerobic GasPak container (BD Biosciences). Plates were returned to the anaerobic chamber for cell harvesting and placement into RNAprotect™. All samples were stored at -80°C until used for RNA-sequencing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNeasy™ RNA purification kit (Qiagen) was used per the manufacturer’s instructions. Total RNA samples were treated with DNase I (Invitrogen). The level of ribosomal RNA present in total RNA samples was reduced prior to library construction using the Ribo-Zero™ Gram Positive Bacteria rRNA Removal Kit (Epicentre Technologies, Madison, WI).
RNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads were aligned to the reference genome using Bowtie2 v. 2.1.0
The resulting sorted aligned reads were analyzed with Cufflinks to measure the relative abundance.The expression of each transcript was quantified as FPKM. Data normalization and differential expression (DE) analysis were done using the DESeq package from Bioconductor. A cutoff false discovery rate adjusted p-value (or q-value) of less than 0.05 was used to select significant DE genes
Genome_build: NC_013316.1
|
| |
|
| Submission date |
May 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Sean Daugherty |
| E-mail(s) |
sdaugherty@som.umaryland.edu
|
| Phone |
410-706-8012
|
| Organization name |
University of Maryland School of Medicine
|
| Department |
Institute for Genome Sciences
|
| Street address |
801 W. Baltimore St.
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21201 |
| Country |
USA |
| |
|
| Platform ID |
GPL20211 |
| Series (1) |
| GSE69001 |
Type IV pili promote early biofilm formation by Clostridium difficile |
|
| Relations |
| BioSample |
SAMN03658416 |
| SRA |
SRX1032543 |
