|
| Status |
Public on May 31, 2019 |
| Title |
EU-RNA rep2 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Xenopus laevis |
| Characteristics |
developmental stage: Mid-blastula transition (MBT), ~ 9 hpf
fraction: EU labeled RNA
|
| Treatment protocol |
Embryos were microinjected with 10 nl of 50 mM 5-EU at 1-cell stage.
|
| Growth protocol |
Embryos were kept at room temperature in 0.1x Marc’s Modified Ringer’s buffer (MMR: 100 mM HEPES pH 7.8, 2 mM EDTA, 2 M NaCl, 40 mM KCl, 20 mM MgCl2, and 40 mM CaCl2).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from normal and EU-microinjected embryos using RNeasy Mini Kit (Qiagen). Nascent EU-RNA was biotinylated by incubating with 25 µM biotin-azide, 100 mM Tris-HCl pH 8.5, 1 mM CuSO4, and 100 mM ascorbic acid for 30 min at room temperature. The biotinylated EU-RNA was pulled down with magnetic streptavidin beads following the instructions provided by the Click-iTTM Nascent RNA Capture Kit (Invitrogen, C10365).
cDNA libraries were prepared from purified EU-RNA (from EU-injected embryos) or total RNA (from normal embryos) following the instructions from the Ovation® Human FFPE RNA-Seq Library Systems (NuGEN) .
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Nascent RNA only
EU2
|
| Data processing |
Base calling was performed by Real-Time Analysis (RTA) version 2.4.11 in Illumina NextSeq 500 system.
The base call (BCL) files were converted to FASTQ files using Illumina bcl2fastq2 Conversion Software v2.20.
Fastq files were aligned to transcripts annotated in Xenopus laevis genome build 9.2 (xenbase.org) using salmon v0.12.0 (Patro et al., 2017) yeilding raw counts per transcript.
Genome_build: Xenopus laevis genome build 9.2 (xenbase.org)
Supplementary_files_format_and_content: Raw counts per transcript, comma separated values text file
|
| |
|
| Submission date |
May 30, 2019 |
| Last update date |
May 31, 2019 |
| Contact name |
Matthew Good |
| E-mail(s) |
mattgood@pennmedicine.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Cell and Developmental Biology
|
| Street address |
421 Curie Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL21248 |
| Series (1) |
| GSE131962 |
Nascent transcriptome of Xenopus laevis embryos at mid-blastula transition (MBT) stages |
|
| Relations |
| BioSample |
SAMN11892991 |
| SRA |
SRX5933459 |
