title: GSM2061028: 3mo RetFloxEGFP-NaV1.8Cre_F_L345_B; Mus musculus; RNA-Seq
library_construction_protocol: 1) Dissect the appropriate level DRGs from the mice carrying a fluorescence reporter of interest, dissociate according to the age appropriate DRG dissociation procedures described on the GUDMAP website under the Jain Group protocols. 2) Add 7-AAD at 1ug/ml concentration, if needed, 10min before sorting to estimate viability and estimating gating parameters (also use fluorescence negative set to establish compensation controls and settings to recover populations of interest). We have had good success using MoFlo, Sony or AriaII, at 100um nozzle size, and 30psi. 3) Collect directly in 750ul TRIzol-LS (Life Technologies, cat# 10296-010). Can store at -80°C or continue with RNA isolation. The RNA was extracted according to M Southard-Smith's RNA isolation procedure from flow sorted cells found on the GUDMAP website (Southard-Smith Group protocols). After Trizol extraction the RNA was purified with Qiagen RNeasy micro kit and then stored at -80°C until further processing for QC and library preparation for RNA sequencing. 4) Following QC/QA on the Nanodrop and Bioanalyzer 'pico' chip the RNA concentration was adjusted to use a total of 10ng in 9ul unless indicated otherwise for library preparation and RNA sequencing. 1) 10ng total RNA (in 9Âµl) was reverse transcribed and amplified using the SMARTerÂ® Ultraâ„¢ Low Input RNA for IlluminaÂ® Sequencing - HV (Clontech, cat# 634828) according to the manufacturerâ€™s instructions. 2) The cDNA was sheared for 3 minutes with the Covaris ultra-sonicator. 3) The libraries were prepared using VeraSeqâ„¢ ULtra DNA Polymerase (Enzymatics, cat# P7520L) for 12 cycles. PCR conditions were 98Â°C for 30s; 12 cycles of 98Â°C for 10s, 64Â°C for 30s, 72Â°C for 30s; 72Â°C for 5 minutes and then hold at 4Â°C. The forward primer used was: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT. The reverse primer contains the unique reverse complement of the respective index nucleotides. The sequence of the reverse primer where "N" denotes the reverse complement of 7 nucleotides of the index is CAAGCAGAAGACGGCATACGAGATNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGA. Refer to the details of the BAM file for the sequence of the index used.
identifiers.namespace: GEO
identifiers.id: GSM2061028