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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 01, 2016 |
| Title |
RNA-seq_24h_rep1 |
| Sample type |
SRA |
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| Source name |
Erythroid progenitors_RNA-seq_24h
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developemental age: E14.5
tissue: fetal liver
cell type: Erythroid progenitors
cultured in: erythropoietin-containing media for 24hrs
molecule subtype: total RNA minus rRNA
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| 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.
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| 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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
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
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| Submission date |
Jun 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Juan R Alvarez-Dominguez |
| Organization name |
Harvard University
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| Department |
Stem Cell and Regenerative Biology
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| Street address |
7 Divinity Ave
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE83823 |
Widespread and dynamic translational control of red blood cell development |
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| Relations |
| BioSample |
SAMN05301573 |
| SRA |
SRX1883495 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2219152_RNA-seq_24h_rep1_neg.bw |
31.1 Mb |
(ftp)(http) |
BW |
| GSM2219152_RNA-seq_24h_rep1_pos.bw |
30.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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