 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 01, 2016 |
| Title |
Ribo-seq_33h_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Erythroid progenitors_Ribo-seq_33h
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developemental age: E14.5
tissue: fetal liver
cell type: Erythroid progenitors
cultured in: erythropoietin-containing media for 33hrs
molecule subtype: ribosome-protected RNA
|
| Growth protocol |
Erythroid progenitors were purified from E14.5 mouse fetal livers by magnetic-assisted cell sorting (Flygare et al., 2011) and cultured for 48hrs in erythropoietin-containing media to induce terminal proliferation and differentiation (Zhang et al., 2003). Samples were collected at 0, 24, 33, and 48 hours and split for RNA-seq and Ribo-seq analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ribo-seq was conducted following published protocols (Ingolia et al., 2012). 50 million cells were harvested at each differentiation time point and no cycloheximide was used. For RNA-seq, total RNA was isolated using the miRNeasy kit (QIAGEN) according to the manufacturer’s instructions. Ribosomal RNA was depleted from 4ug total RNA using the Ribo-Zero Gold Kit (Epicentre, Madison, WI).
Strand-specific cDNA libraries were prepared using a dUTP marking protocol (Borodina et al., 2011) and sequenced on an Illumina HiSeq2000 platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
library strategy: Ribo-seq
Ribo-seq reads were quality-filtered with the fastq_quality_trimmer (“-t 20” parameter) tool of the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) and clipped to remove 3’ linkers using the fastx_clipper tool, discarding non-clipped reads or reads <25nt after linker clipping (“-l 25 -c” parameters). Reads were then aligned to reference rRNA, tRNA, and mitochondrial DNA sequences using Bowtie v.1.1.1 with default parameters and “--seedlen=23”to remove these RNAs species. Finally, we used fastx_trimmer to remove the first 5’ nucleotide from each read, as it often reflects an untemplated addition during cDNA generation. Pre-processed RFPs were then aligned to a reference transcriptome composed of Ensembl v.67 annotations supplemented with erythroid lncRNA and TUCP annotations (Alvarez-Dominguez et al., 2014) using TopHat v2.0.13 with default parameters and “--min-anchor 5 --segment-length 20 --read-mismatches 1 --no-novel-juncs”. For RNA-seq, reads aligned by Bowtie to rRNA, tRNA, or mitochondrial DNA were discarded. As with Ribo-seq analysis, the first 5’ nucleotide was trimmed from each read using fastx_trimmer, reads were subsequently aligned to a composite reference transcriptome using TopHat.
Genome_build: mm9
Supplementary_files_format_and_content: mapped reads in bigwig
|
| |
|
| Submission date |
Jun 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Juan R Alvarez-Dominguez |
| Organization name |
Harvard University
|
| Department |
Stem Cell and Regenerative Biology
|
| Street address |
7 Divinity Ave
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE83823 |
Widespread and dynamic translational control of red blood cell development |
|
| Relations |
| BioSample |
SAMN05301568 |
| SRA |
SRX1883490 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2219147_Ribo-seq_33h_rep2_neg.bw |
7.8 Mb |
(ftp)(http) |
BW |
| GSM2219147_Ribo-seq_33h_rep2_pos.bw |
7.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
