GSM1542156: E14.5 DRG (3ng, high volume); Mus musculus; RNA-Seq
1)  DRGs from eighteen E14.5 embryos were lysed in TRIzol-LS (Life Technologies, #10296-010).  2. 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).  3. Following QC/QA on the Nanodrop and Bioanalyzer 'pico' chip the RNA concentration was adjusted to 100ng/ul and used as a master stock. This was used to make further dilutions as needed for the RNAseq experiments. library_construction_protocol : 1) 3ng, 10ng or 30ng total RNA (in 9µl) was reverse transcribed and amplified using the SMARTer® Ultra™ Low Input RNA for Illumina® Sequencing - HV (Clontech, cat# 634828) according to the manufacturer’s instructions.  2) The cDNA was sheared for 3 minutes with the Covaris ultra-sonicator.   3) The libraries were prepared using VeraSeq™ ULtra DNA Polymerase (Enzymatics, cat# P7520L) for 12 cycles.  PCR conditions were 98°C for 30s; 12 cycles of 98°C for 10s, 64°C for 30s, 72°C for 30s; 72°C for 5 minutes and then hold at 4°C.  The forward primer used was: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT. The reverse primer with the indexes can be viewed on the GUDMAP website (Jain Group protocols).