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| Status |
Public on Sep 01, 2013 |
| Title |
Mouse digit 0hr |
| Sample type |
SRA |
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| Source name |
mouse tissue
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| Organism |
Mus musculus |
| Characteristics |
tissue: hindfoot digit
strain: C57/BL6
time course: 0hr
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| Treatment protocol |
For Mouse: Following induction of surgical plane anesthesia with 1-2% inhaled isoflurane (assessed by intact breathing, intact femoral pulses bilaterally, and lack of response to tail and foot stimulus), adult, 6 to 8 week old C57/BL6 mice (Jackson Laboratories, Bar Harbor, ME) were subjected to aseptic, mid-second phalanx amputation of the third digit of each hindfoot. Post surgical buprenorphine and baytril were administered for 3 days post-surgery for analgesia and antibiotic prophylaxis, respectively. Animals were sacrificed and the distal portion of each amputated digit (distal to the proximal joint) was harvested into RNAlater solution (Ambion, Austin, TX) and immediately flash frozen in liquid nitrogen. Time points of harvest included 0 hours, 3 hours, 6 hours, 12 hours, 1 day, 5 days, 10 days, and 14 days. Following transfer of each sample overnight on dry ice to the Morgridge Institute for Research (Madison, WI), RNA from each sample was isolated using Trizol (Invitrogen, Carlsbad, CA) and treated with Dnase I. Then the RNA was purified by extraction with phenol:chloroform.
For Axolotl: For all surgical procedures, the animals were anesthetized with 0.5-1 g/L Tricaine (MS-222, Sigma, St. Louis, MO) until they were unresponsive to a tail pinch stimulus.We amputated juvenile limbs at the mid-stylopod level. Tissue was harvested as described in Figure 1 at 0 hours, 3 hours, 6 hours, 12 hours, 1 day, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, and 28 days. Note that for the early time points, some tissue was harvested proximal to the original amputation plane owing to the small amount of regenerative tissue distal to the amputation plane at the time of harvesting. In all cases the harvested tissues were stored in RNAlater (Qiagen, Valencia, CA) at 4OC until the RNA was purified using the RNeasy purification kit from Qiagen.
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| Growth protocol |
For Mouse: Mice were housed at the animal facility at the University of Pittsburgh
For Axolotl: The animals were housed in 40% Holtfreter’s salts, kept at 16-18O C with a pH range of 7.0-7.4.
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| Extracted molecule |
total RNA |
| Extraction protocol |
We linearly amplified axolotl polyA+ RNAs using a modified T7 amplification method (Sengupta et al., 2010) resulting in cDNA. Subsequent steps followed the Illumina Paired End (PE) preparation kit (PE-102-1001, Illumina, San Diego, CA). After the Illumina PE adapters were ligated, 150-250 bp DNA fragments were isolated via gel electrophoresis. Then ten cycles of polymerase chain reaction (PCR) were performed to amplify the selected fragments using the Illumina supplied PCR primers and protocol. The sample was quantitated with the Invitrogen Qubit fluorometer (Q32857). Samples were loaded on the flowcell cluster station at a concentration of 8pM. The Genome Analyzer II Paired End recipe was used. Some runs were single-read runs, while some runs were paired-end.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
For data analysis, paired-end reads were treated as two separate single-end reads. After the sequencing was complete, the data were processed by Illumina Pipeline software for quality analysis. For transcript quantification and differential analyses, we used an axolotl contig set assembled with MIRA (Chevreux et al., 2004) from a combination of Sanger and 454 EST sequences. Details of the assembly process are provided in Supplemental Experimental Procedures. We used BLAST to align the contigs against human RefSeq (Pruitt et al., 2009) RNA and protein sequences, taking the best BLAST hit with e-value less than 10-5 as the most closely related human homolog for each contig. RSEM (Li and Dewey, 2011) was used to estimate the relative abundances and expected read counts for the contigs. Contigs mapping to rRNA transcripts were removed and abundances were renormalized. The expected read counts for contigs mapping to the same homologous human transcript were summed to give abundances. Read counts for transcripts belonging to the same gene were summed to give human gene-level abundances.
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| Submission date |
Dec 12, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeff Nie |
| Organization name |
Mayo Clinic
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| Street address |
200 First St. SW
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| City |
Rochester |
| State/province |
MN |
| ZIP/Postal code |
55905 |
| Country |
USA |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE34394 |
Three Distinct Phases of Regeneration-Specific Gene Expression in the Axolotl Blastema |
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| Relations |
| SRA |
SRX111253 |
| BioSample |
SAMN00765331 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM848190_mouse_digit_TC_gene_tpm_0h.txt.gz |
217.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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