Whole blood RNA-seq gene expression in ALS cases versus healthy controls
When total RNA samples (90 ng) met the criteria RIN less than 5.5 or DV200 in the range of 50 to ~75%, they were depleted of rRNA using the NEBNext Globin & rRNA Depletion Kit (catalog no. E7750X, New England Biolabs, Ipswich, MA). This was the protocol adopted for 261 ALS and 126 control samples. rRNA-depleted RNA samples were then fragmented for 5-10 min based on RIN of the input RNA. Fragments were then copied into first-strand cDNA by reverse transcription with random primers, and 3' ends were adenylated and ligated to adapters. The products were amplified by 16 PCR cycles to generate the final cDNA library. Library preparation was performed using xGen Broad-Range RNA Library Preparation Kit (catalog no. 10010145, Integrated DNA Technologies, Coralville, IA) and xGen Normalase UDI Primers (catalog no. 10009795, 10009800, 10009811, 10009812, Integrated DNA Technologies). RNA-seq analysis adjusted for RNA processing method.