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Sample GSM2303118 Query DataSets for GSM2303118
Status Public on Jan 20, 2017
Title FL_RPF 3
Sample type SRA
Source name Mouse
Organism Mus musculus
Characteristics cell type: microdissected forelimb buds (FL) at E11.5 embryos
genotype: FVB
Treatment protocol Cells of mesodermal lineage derived from the primitive streak were isolated by Fluorescence Activated Cell Sorting (FACS) at E11.5 embryos. Neural tubes (NT) and forelimb buds (FL) were microdissected from E11.5 embryos.
Growth protocol Mice were housed under a 12 hr light/dark cycle with free access to food and water. All animal work was performed in accordance with protocols approved by Stanford University’s Administrative Panel on Laboratory Animal Care.
Extracted molecule total RNA
Extraction protocol For Ribo-Seq, the cell pellet was re-suspended in 200 μl of cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1 mM DTT, 1% Triton X-100, 8% glycerol, 20 U/ml TURBO™ DNase (Ambion, AM1907)) and incubated for 30 min at 4°C with occasional vortexing. The lysate was clarified by sequential centrifugation for 5 min at 1800 x g and 10000 x g at 4°C to remove nuclei and mitochondria. The lysate was then treated with RNase I (Ambion, AM2294) for 30 min at RT to digest mRNAs not protected by the ribosome. The digestion was stopped by adding 4.5 μl of SUPERaseIn™ RNase Inhibitor (20 U/μl, Ambion AM2696). Lysate was then loaded onto a 1 M sucrose cushion. Ribosomes were pelleted by ultracentrifugation at 70000 rpm for 4 hr at 4°C by TLA120.2 rotor. The pellet was re-suspended in Trizol (Invitrogen, 15596) and RNAs were extracted by following manufacturer’s protocol. For RNA-Seq, after centrifugation, the cell pellet was re-suspended in Trizol (Invitrogen, 15596) for RNA extraction. RNA was extracted and polyA mRNA was isolated using Oligotex mRNA Mini Kit (Qiagen, 70022) following manufacturer’s protocol. Purified mRNAs were fragmented in alkaline fragmentation buffer (100 mM NaCO3 pH9.2, 2 mM EDTA).
Briefly, ribosome protected fragments (RPFs) and fragmented RNAs were loaded onto a 15% urea gel. 28-31 nt RPFs and 30-50 nt fragmented RNAs were excised from the gel for Ribo-Seq and RNA-Seq respectively. RNAs were eluted, dephosphorylated by PNK (NEB, M0201S), and ligated to the miRNA Cloning linker (NEB, S1315S) by T4 RNA Ligase2 truncated K227Q (NEB, M0242S). Ligated RNA was gel purified and reverse transcribed by Superscript III (Invitrogen, 18080). Gel purified cDNAs were circularized by Circligase (Epicentra, CL4111K) and rRNA sequence were subtracted using biotinylated oligos. Amplification was done using Phusion High Fidelity DNA Polymerase (NEB, M0530S). PCR amplification was performed for 10-15 cycles and products were loaded onto non-denaturing 8% PAGE gel. DNA fragment were purified for Illumina sequencing.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
Description Ribosome-Protected Fragments (RPFs)
Data processing Sequencing reads were parsed by cutadapt to remove the 3’ adapter sequence and reads with good sequencing qualities (phred quality score>33) were kept.
A layered alignment employing Bowtie 2 was performed to first discard reads mapping to rRNA, tRNA or snRNA sequences. Non-rRNA/tRNA/snRNA reads were then aligned against the canonical isoform of UCSC known gene transcripts (mm10).
Mapped footprints were assigned to a specific position based on the A sites. The position of A site in relative to the 5’ end of each read is calculated as follows: 29-30 nt: +15; 31-33 nt: +16 and 34-35 nt: +17.
For each gene, the total number of ribosome footprints mapping to the CDS excluding the first 15 or last 5 codons were counted. The density of ribosome footprints was calculated as Reads Per Kilobase per Million mapped reads (RPKM). Genes having ≥5 RPKM were kept for further analysis.
Genome_build: mm10
Supplementary_files_format_and_content: Text files include normalized read counts (DESeq) for each analyzed protein-coding genes
Submission date Sep 06, 2016
Last update date May 15, 2019
Contact name Zhen Shi
Organization name Stanford
Street address 279 Campus Drive
City Stanford
State/province California
ZIP/Postal code 94305
Country USA
Platform ID GPL19057
Series (1)
GSE86467 Pervasive translational regulation of the cell Q1 signalling circuitry underlies mammalian development
BioSample SAMN05730962
SRA SRX2140594

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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