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Status |
Public on Aug 26, 2016 |
Title |
n3.11 |
Sample type |
SRA |
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Source name |
L5 DRG intact single cell
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Organism |
Mus musculus |
Characteristics |
strain: CAST/Eij male X C57BL/6 female tissue: dorsal root ganglion
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Extracted molecule |
polyA RNA |
Extraction protocol |
The experiment was performed on two month old adult CAST/Eij ♂ cross C57BL/6♀F1 mice. All surgical procedures were performed under general anesthesia with intraperitoneal injections using a mixture of ketamine(100mg/kg) and xylazine(10mg/kg). For the sciatic nerve transection (axotomy), the right side sciatic nerve was exposed at the mid thigh level and sectioned distally. The wound was sutured in two layers, and the animals were allowed to recover. After 3 days of the surgery, animals were euthanased by CO2 and decapitated, L5 DRG from both ipsilateral and contralateral side were dissected and dissociated into single cell. Single DRG soma was manually picked in the lowest possible volume (preferably ≤0.5μl, possibly 0.3μl) using a micro capillary pipette into a 0.2-ml thin-walled PCR tube contains 4μl smart-seq2 lysis buffer. Cell sizes were measured and recorded during the picking. Except for the enzyme dissociation, all the dissection procedure were performed on ice in order to reduce RNA degradation. Whole transcriptome amplification in tubes were performed following Smart-seq2 protocol (Picelli, Faridani et al. 2014) with minor edition. Briefly, the nuclei were lysised and ploy-A RNA was reverse transcribed by superscript III reverse transcription enzyme using a template switch fashion. cDNA were then amplified by KAPA polymerase for 18 PCR cycles. After purification, 0.2ng cDNA were used for Nextera tagmentation and library construction. ERCC RNA spike-in Mix (Ambion, Life Technologies) was added to the lysis reaction and processed in parallel with ploy-A RNA. All libraries were sequenced 50bp single end on illumina Hi-seq2000 pipeline.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
DRG_Nor_51
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Data processing |
CASAVA 1.8.2 was used to separate out the data for each single cell by using unique barcode combinations from the Nextera XT preparation and to generate fastq files. Raw reads past quality check were mapped by bowtie2, gene expression level was quantified as transcripts per million reads (TPM) using RSEM to known exons using Refseq gene annotation downloaded from UCSC genome browser. Ribosomal RNA annotations were removed from reference file before gene expression quantification. Genome_build: mm9 Supplementary_files_format_and_content: In order to reduce technical noise, We filtered away genes with greater than 20% samples with TPM<10. Then we did log2 transform on the remaining data.
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Submission date |
Jul 28, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Ganlu Hu |
E-mail(s) |
huganlu@gmail.com
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Phone |
+8613636618854
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Organization name |
University of California, Los Angeles
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Department |
human genetics
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Lab |
Guoping Fan lab
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Street address |
695 Charles E Young Dr. South
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City |
Los Angeles |
State/province |
CA |
ZIP/Postal code |
90095 |
Country |
USA |
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Platform ID |
GPL13112 |
Series (1) |
GSE71453 |
Single cell RNA-seq analysis of sensory neurons reveal diverse injury responses after sciatic nerve transection |
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Relations |
BioSample |
SAMN03940903 |
SRA |
SRX1122458 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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