|
| Status |
Public on Mar 24, 2023 |
| Title |
S.islandicus.REY15A.E234.Cappable-seq.replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
microbial cells
|
| Organism |
Sulfolobus islandicus REY15A |
| Characteristics |
genotype: E234 (pyrEF, lacS)
cell type: microbial cells
|
| Growth protocol |
Strains were grown in Brock medium supplemented with 0.2% sucrose, 0.2% tryptone, and 20 µg/ml uracil at 75 °C in Erlenmeyer shake flasks at 150 rpm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
15 ml cell culture was rapidly mixed with 30 ml pre-cooled RNAprotect Bacteria Reagent (Qiagen) placed in an ice bath. Cells were harvested by centrifugation (5 min at 4000 x g at 4 ºC). Pellets were immediately subjected to RNA isolation using the mirVana miRNA isolation kit (Ambion) with an initial resuspension buffer volume of 200 µl following the protocol for small RNAs (20-200 nt length).
Library preparation and deep sequencing was conducted at Vertis Biotechnologie (Germany). In brief, 5’-triphosporylated RNA was capped with 3’-desthiobiotin-TEG-GTP (NEB)) using the Vaccinia virus Capping enzyme (NEB) and biotinylated RNA species were subsequently enriched by affinity purification using streptavidin beads yielding 0.6 to 1.3% of the sRNA preparation. The eluted RNA was poly-adenylated using E. coli Poly(A) polymerase and 5’-ends were converted to mono-phosphates by incubation with RNA 5’ Pyrophosphohydrolase (NEB). Subsequently, an RNA adapter was ligated to the newly formed 5'-monophosphate structures. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M- MLV reverse transcriptase at 42 ºC. The resulting cDNA was finally PCR-amplified (12 cycles) with TruSeq Dual Index sequencing primers (Illumina).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
E234_r1
|
| Data processing |
library strategy: Cappable-seq
In order to remove poly(A)-tails and adaptors, we trimmed the reads using the program Cutadapt in two rounds with the following settings to prevent trimming of naturally occurring A-rich RNAs due to the low GC-content of the S. islandicus REY15A genome: (i) -a “A{15}” -e 0 -m 15 to remove all poly(A) stretches of at least 15 nt length plus downstream regions and (ii) -a “A{15}”X -e 0 -O 5 to terminal shorter poly(A) stretches of minimum 5 nt length.
Trimmed and untrimmed reads were split into separate fastq files using awk.
Both fastq files were aligned to the S. islandicus REY15A genome using bowtie v1.2.2 61 (parameters -v 1 -m 1 –-best –-strata -S) with untrimmed 75 nt reads shortened to 71 nt (-3 4)
The bam file output was merged, sorted and indexed using SAMtools
Read 5'-end coverage per strand was calculated using bedtools genomecov
Assembly: NC_017276.1
Supplementary files format and content: Big wig file with read 5'-end coverage per strand
|
| |
|
| Submission date |
Feb 24, 2023 |
| Last update date |
Mar 24, 2023 |
| Contact name |
Finn Werner |
| E-mail(s) |
[email protected]
|
| Organization name |
University College London
|
| Department |
Institute of Structural and Molecular Biology
|
| Street address |
Gower Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL33183 |
| Series (2) |
| GSE226022 |
Cappable-seq data for Sulfolobus islandicus REY15A and cbp1 deletion strains |
| GSE226026 |
Cbp1-Cren7 chromatinization of CRISPR arrays favours transcription from CRISPR leaders over cryptic promoters. |
|
| Relations |
| BioSample |
SAMN33434657 |
| SRA |
SRX19494937 |
