|
| Status |
Public on Sep 23, 2023 |
| Title |
Rnase R treated-Chronic infection without CNS pathology (CP-)-BH-2 |
| Sample type |
SRA |
| |
|
| Source name |
Brain
|
| Organism |
Macaca nemestrina |
| Characteristics |
tissue: Brain
treatment: Rnase R treated-Chronic infection without CNS pathology (CP-)-BH-2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
BH RNA was extracted by adding Trizol (Thermo Fisher15596018) and homogenizing tissues with Lysing Matrix D (MP Biomedicals116913100) in a benchtop homogenizer (FastPrep-24, MP Biomedicals) at 4.0 m/s for 20 seconds. After homogenization, the supernatant was collected, and RNA was isolated by miRNeasy Mini Kit solutions (Qiagen217004) and Zymo-Spin IIICG Columns (Zymo Research C1006-50-G) following the manufacturer's instructions.
1 µg of BH RNAs was incubated with 1 U/µg RNase R (Lucigen RNR07250) at 37°C for 30 min. RNase R-treated RNAs were then re-isolated by RNA Clean & Concentrator™-5 (Zymo Research R1014) according to the manufacturer’s protocol. 100 ng of total BH RNA with and without RNase R treatment was then used to construct cDNA libraries by Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Illumina 20072063). A Ribo-Zero Plus kit was used to deplete ribosomal RNA in these samples, and library construction was then done per manufacturer’s protocol. Indexes were attached using IDT for Illumina RNA UD Indexes according to the manufacturer’s protocol. The yield and size distribution of the total RNA libraries were assessed using the Fragment Bioanalyzer™ system with DNA 1000 chip (Agilent 5067-1505). Multiplexed libraries were equally pooled to 1nM and sequenced with the NovaSeq 6000 system (Illumina) and NovaSeq 6000 S2 Reagent Kit v1.5 (300 cycles) (Illumina 20028314).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
circRNA_CiRISeq_Yiyao9_R1R2_mmul_circRNA_041121_final
circRNA_circExp-raw_counts
|
| Data processing |
The raw reads were first trimmed from contaminating adapter sequences using cutadapt software. The trimmed and size-selected (>15 nt) reads were then aligned using Bowtie2 (default settings) to the manually curated M. mulatta mRNA reference containing a single (main) transcript per each gene (designated with a gene symbol, e.g., GAPDH). All reads which did not align to the above mRNA were mapped to the combined M. mulatta cDNA and ncRNA references from Ensembl (designated with transcript ID, e.g., ENSMMUT00000047080.3). The numbers of reads mapped to each transcript were extracted using eXpress software based on a previous publication (Roberts and Pachter, 2013).
circRNA identification by Sequential Alignment (CiRISeqA): All reads aligned to the M. mulatta combined cDNA and ncRNA references from Ensembl were discarded. The remaining reads were additionally filtered from non-circular RNA sequences by removing all reads mapped to "non-modified" M. mulatta CircAtlas v.2 references (downloaded from: http://circatlas.biols.ac.cn/) using the same Bowtie settings. Next, the remaining reads were aligned to "duplicated" CircAtlas v.2 reference transcripts that correspond to sequences mapped to the junction regions of circRNAs. The numbers of reads mapped to each duplicated RNA reference were extracted using eXpress software based on a previous publication (Roberts and Pachter, 2013). See also Supplementary Figure 2.
circRNA identification by circExplorer2: The genome and gene annotations for M. mulatta were obtained from UCSC (BCM Mmul_8.0.1/rheMac8). First, raw files were aligned to the genome using STAR (chimSegmentMin 10), and the chimeric junction files were used to count circRNAs using circExplorer2. circRNAs identified by both our custom pipeline (above) and circExplorer2 were included in the following analysis.
Assembly: M. mulatta
Supplementary files format and content: Excel files contains all mapped reads
|
| |
|
| Submission date |
Jul 10, 2023 |
| Last update date |
Sep 23, 2023 |
| Contact name |
Kenneth W. Witwer |
| E-mail(s) |
kwitwer1@jhmi.edu
|
| Phone |
4109559770
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Department of Molecular and Comparative Pathobiology
|
| Street address |
733 N. Broadway
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL33261 |
| Series (2) |
| GSE236931 |
Total RNA profiles of brain tissue in simian immunodeficiency virus (SIV) infection and SIV-related central nervous system pathology |
| GSE236937 |
RNA profiles of brain tissue |
|
| Relations |
| BioSample |
SAMN36396148 |
| SRA |
SRX20970251 |
