|
| Status |
Public on Jul 31, 2020 |
| Title |
RsPhoto_RppH_Plus_Rep3 |
| Sample type |
SRA |
| |
|
| Source name |
RsPhoto_RppH_Plus
|
| Organism |
Cereibacter sphaeroides 2.4.1 |
| Characteristics |
genotype/variation: RppH+
growth condition: photosynthetic
growth phase: exponential
|
| Growth protocol |
Three replicates of R. sphaeroides 2.4.1 cultures were inoculated from an overnight culture into Sistrom’s minimal medium (14) and grown at 30°C. Aerobic cultures were grown in roux bottles and sparged with 1% CO2, 30% O2, and 69% N2 while anaerobic cultures were sparged with 5% CO2 and 95% N2 while exposed to an incandescent light with an intensity of ~10 W/m2 as previously described (15). Aerobic cultures were grown until mid-log phase (OD600 of ~0.3-0.4) and anaerobic cultures were collected at an OD600 of ~0.7-0.9 to achieve similar cell densities between the two growth conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using a hot phenol method
The isolated RNA was split into two equal groups and one group was treated with the enzyme RppH (17) (NEB, M0356S) at 37°C for 2 hours to remove the outer two phosphate groups from the 5ʹ end of the non-processed RNA species (RppH+ sample). The other group is untreated (no incubation with RppH) and thus the non-processed RNA species retain a tri-phosphate at the 5ʹ end of these molecules (RppH- sample). Both groups are treated with NaIO4 for 1.5 hours at 4°C in the dark to inactivate the 3ʹ end of the RNA transcripts and reduce the production of side products during the subsequent ligation of amplification primers (18). The sequencing adapter with desired index barcode sequence is ligated to the 5ʹ end of RNA species with a single phosphate group (Table 1). Note that RNA species with tri-phosphate groups at the 5ʹ end of the RNA are not substrates for ligation and thus will form a product with this sequencing adapter. The ligated RNA is purified using SPRI beads (Beckman, B23317), eluted in water, and quantitated using a NanoDrop 2000. Both the RppH+ and RppH- samples are subjected to rRNA depletion using the RiboZero kit (Epicentre, MRZB12424) following standard protocols and then reverse transcribed using a random primer attached to the second Illumina sequencing adapter (Table 1) following manufacturer’s recommendations. PCR is used to amplify cDNA species in both the RppH- and RppH+ samples that contain both Illumina sequencing adapters using a KAPA Hi-Fi PCR-read mix (KAPA Biosystems KK2601) following manufacturer’s protocol. The PCR products are cleaned using SPRI beads twice and resuspended in a final volume of 20 µL. The TSS-seq library is quantified using a 2100 Bioanalyzer (Agilent) and identifying fragments ~130 bp.
The samples were sequenced on an Illumina HiSeq 2500
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Rhodobacter sphaeroides Transcription Start Site sequencing sample grown under photosynthetic conditions (1% CO2 and 99% O2). RppH Plus. Biological repliate 3
|
| Data processing |
The resulting FASTQ files are split using the index barcode sequences to separate the sequences for the RppH- and RppH+ samples using fastx_barcode_splitter.pl version 0.0.13.2 (www.hannonlab.cshl.edu/fastx_toolkit).
The sequences are then trimmed to remove any remaining adapter-derived bases using Trimmomatic version 0.3 (HEADCROP of 6, MINLEN of 25) (19).
The trimmed sequence reads are aligned to the R. sphaeroides genome (GCF_000012905.2, Assembly ASM1290v2) using Bowtie2 version 2.3.5.1 (20), allowing for one mismatch. The aligned Bowtie2 file is then further processed with Picard tools version 2.10.0 (https://broadinstitute.github.io/picard/) and samtools (21).
The bedtools version 2.27.0 genomeCov command (https://bedtools.readthedocs.io/en/latest/) is used to map genomic locations that corresponds to the position of the first base in each aligned read, which is defined as the transcription start site. A pseudocount of 1 was added to all TSS read values to prevent dividing by zero and edgeR version 3.10 (22) was used to identify locations with a statistically significant increase in read abundance in the RppH+ strain compared to the RppH- strain.
Locations with a significant increase in read count in the RppH+ sample (FDR ≤ 0.05) were retained and TSSs were associated with genes downstream within 350 bp of the translation start site.
Genome_build: GCF_000012905.2, Assembly ASM1290v2
Supplementary_files_format_and_content: Bedtools GenomeCov mapped read value to each location in the genome; TSS locations
|
| |
|
| Submission date |
May 20, 2020 |
| Last update date |
Jul 31, 2020 |
| Contact name |
Kevin S Myers |
| E-mail(s) |
kmyers2@wisc.edu
|
| Organization name |
University of Wisconsin - Madison
|
| Department |
Great Lakes Bioenergy Research Center
|
| Street address |
5127 WEI, 1552 University Ave
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53726 |
| Country |
USA |
| |
|
| Platform ID |
GPL18842 |
| Series (1) |
| GSE150944 |
Genome-wide Identification of Transcription Start Sites in Two Alphaproteobacteria |
|
| Relations |
| BioSample |
SAMN13935673 |
| SRA |
SRX7639959 |
