|
| Status |
Public on May 25, 2019 |
| Title |
D14-400966 |
| Sample type |
SRA |
| |
|
| Source name |
regenerating_non-pole_Isolated planarian cell
|
| Organism |
Schmidtea mediterranea |
| Characteristics |
strain: asexual CIW4
ploidy: 2N (non-dividing)
uninjured or regenerating animal: regenerating animal 72 hours post transverse amputation
cell type: non-pole muscle cell
|
| Treatment protocol |
Single cell suspensions for each region were stained with Hoechst, and non-dividing single cells were sorted by flow cytometry into 96 well plates
|
| Growth protocol |
Asexual Schmidtea mediterranea strain (CIW4) animals starved 7-14 days before surgical resection of midline tail tip regions, or before transverse tail amputation, then resection of midline posterior blastema regions after 72 hours of regeneration.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were lysed in total cell lysis buffer supplemented with 1% β-mercaptoethanol
cDNA and sequencing libraries were constructed using SmartSeq2.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
muscle cell by qPCR collagen expression
140929Red_D14-400966
processed data file; raw read counts:
ReadcountMaster_uniquegenes2_forGEO_onlySinglecells.txt
processed data file; differential expression analysis:
ppolecandidates_singlecell_PvsNP_modelbygroup_forGEO.txt
|
| Data processing |
Basecalls performed using CASAVA version 1.7.0.
Reads were trimmed using Trimmomatic-0.32 to remove Nextera transposon sequences and low quality 3′ base pairs.
Reads were mapped to dd_Smed_v4 planarian transcriptome assembly using bowtie 1 with --best to generate raw read counts file.
Only cells with a total read count of >10,000 from all genes were included in subsequent analysis. Read counts were normalized to cpm.
Cells with collagen (dd_Smed_v4_702_0_1) cpm <40, troponin (dd_Smed_V4_323_0_2) cpm <80, and with tropomyosin (dd_Smed_v4_436_0_1) cpm of 0 were excluded from analysis
Cells with high AGAT expression were excluded from analysis
Reads for isotigs of contigs (all contig ids that vary only by the final number) were summed.
Reads for mitochondrial and ribosomal RNA (dd_Smed_v4_7_0, and dd_Smed_v4_4_1, mtRNA, rRNA_5s) were discarded.
Differential gene expression analysis was performed between posterior pole cells (11 total) and non-posterior-pole muscle cells (90 total) using SCDE analysis.
Genome_build: dd_Smed_v4
Supplementary_files_format_and_content: Raw read counts: Tab-delineated text file includes unnormalized read counts of contigs for all 152 cells. SCDE analyses: tab-delineated text file generated per Kharchenko et al, 2014 for differential gene expression between posterior pole cells (11 total) and non-posterior-pole muscle cells (90 total) that passing quality filters (per data processing steps delineated above). Fold change is indicated as the maximum likelihood estimate (mle) with a lower (lb) and upper bound (ub) and a conservative estimate (ce, equal to lb (positive), ub (negative) or 0 (lb and ub have different signs)) and statistical significance as signed Z-score (Z) and conservative Z-score (cZ, Z score corrected for multiple hypothesis using Holm procedure).
|
| |
|
| Submission date |
Oct 10, 2018 |
| Last update date |
May 25, 2019 |
| Contact name |
Peter W Reddien |
| Organization name |
Whitehead Institute for Biomedical Research
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL15553 |
| Series (2) |
| GSE121047 |
RNA sequencing of single cells from the midline posterior extremity in planarian Schmidtea mediterranea |
| GSE121048 |
RNA sequencing studies in planarian Schmidtea mediterranea |
|
| Relations |
| BioSample |
SAMN10227395 |
| SRA |
SRX4823281 |
