GSM1834966: s3.11; Mus musculus; RNA-Seq
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.
GEO Accession
GSM1834966