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| Status |
Public on Sep 03, 2018 |
| Title |
rna_seq_ko_plus_r2 |
| Sample type |
SRA |
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| Source name |
Fetal liver
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| Organism |
Mus musculus |
| Characteristics |
developmental stage: E14.5
tissue: liver
strain: C57BL/6
cell type: Primary basophilic erythroblasts
genotype: Hri-/-
condition: iron sufficiency
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For Ribo-seq, sorted erythroblasts were washed twice with cold phosphate-buffered saline and then treated with cycloheximde (100 ug/ml) at 37℃ for 5 min, followed by isolation of ribosome protected fragments (RPFs) using ARTseq-Ribosome Profiling Kit (Illumina). For mRNA-seq, total RNAs were extracted by RNeasy Plus kit (Qiagen) and polyA RNAs were isolated using an Oligotex mRNA kit (Qiagen).
Ribo-seq cDNA libraries were prepared as described (Ingolia et al. 2009, Ingolia, Lareau, and Weissman 2011). Briefly, RPFs were dephosphorylated and linkers were ligated using T4 RNA ligase. RNA samples were then reverse transcribed, circularized and PCR amplified for 12 cycles. PCR products were subjected to gel purification before sequencing. mRNA-seq cDNA libraries were prepared by the MIT BioMicro Center.
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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 |
Fastq files were trimmed by Cutadapt to remove adapters and reads with a base quality score less than 10. Reads with length less than 26 or 9 nucleotides were also discarded for Ribo-seq and mRNA-seq samples, respectively. For Ribo-seq samples, reads containing rRNA and tRNA sequences were further removed using Bowtie2.
After quality control analysis using FastQC, reads were mapped to mouse genome mm10 (UCSC) using STAR aligner with maxima of 2 mismatches and 8 multiple loci.
Quality of Ribo-seq data was examined by the triplet periodicity using RibORF.
The uniquely mapped reads were counted using Htseq. For Ribo-seq data, the first 15 and the last 5 codons were removed from reads counting to avoid the bias of pile up of reads around the start codons and stop codons.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include gene symbol on the first column and the raw read count values on the second column.
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| Submission date |
Sep 02, 2018 |
| Last update date |
Sep 04, 2018 |
| Contact name |
Shuping Zhang |
| Organization name |
Massachusetts Institute of Technology
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| Street address |
77 Massachusetts Avenue
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE119365 |
Iron and Heme Coordinate Erythropoiesis through HRI-Mediated Regulation of Protein Translation and Gene Expression |
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| Relations |
| BioSample |
SAMN09946128 |
| SRA |
SRX4633094 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3371784_rna_seq_ko_plus_r2.txt.gz |
90.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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