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Sample GSM1834954

Query DataSets for GSM1834954

Status

Public on Aug 26, 2016

Title

n3.21

Sample type

SRA

Source name

L5 DRG intact single cell

Organism

Mus musculus

Characteristics

strain: CAST/Eij male X C57BL/6 female
tissue: dorsal root ganglion

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

DRG_Nor_66

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.

Submission date

Jul 28, 2015

Last update date

May 15, 2019

Contact name

Ganlu Hu

E-mail(s)

huganlu@gmail.com

Phone

+8613636618854

Organization name

University of California, Los Angeles