Quantification Method

Sequencing

Human Dorsal Root Ganglia from the L2 lumbar region was extracted from adult female donor samples, and preserved in RNA Later. RNA-seq library preparation was performed using Illumina TruSeq RNA Sample Kit v2. PolyA+ cDNA paired-end sequencing was performed on Illumina NextSeq 500, with individual read length being 75 bp. Tissue sourcing and extraction was performed by Anabios, Inc. Sequencing was performed by Active Motif, Inc.

Quantification

For all sequencing experiments, identical uniform processing guidelines were used as follows. Reference transcriptomes used were Gencode v14 ( based on hg19 ) for human , and Gencode vM4 ( based on mm10 ) for mouse. [1] Tophat v2 was used for mapping reads to the reference genome and transcriptome .[2] Cufflinks was used for quantifying reads : classic FPKM normalization was used. [2] Further, a TMM based re-normalization strategy was used to minimize lab / batch effects to generate a normalizing constant for each experiment. [3] Finally, a set of polyA+, rRNA- gene set was extracted and the TPM was calculated based on the renormalized FPKM. Human and mouse gene orthologs were identified using the Homologene database ( http://www.ncbi.nlm.nih.gov/homologene) . [4]

References