Whole blood RNA-seq gene expression in ALS cases versus healthy controls
When total RNA samples (90 ng) met the criteria RIN greater than and equal to 5.5 and DV200 greater than 75%, mRNA was isolated by polyA purification using the NEBNext Poly(A) mRNA Magnetic Isolation Module (catalog no. E7490L, New England Biolabs). This was the protocol adopted for 161 ALS and 146 control samples. mRNA-purified samples were then fragmented and copied into first-strand cDNA by reverse transcription 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 Prep (catalog no. 10010145, Integrated DNA Technologies), and xGen Normalase UDI Primers (catalog no. 10009795, 10009800, 10009811, 10009812, Integrated DNA Technologies). RNA-seq analysis adjusted for RNA processing method. The quality of all final libraries, both rRNA-depleted and mRNA-purified, was assessed by Qubit dsDNA (Thermo Fisher Scientific) and LabChip (PerkinElmer, Waltham, MA). Samples were pooled and sequenced on an Illumina NovaSeq S4 using 150 bp paired-end reads (Illumina, San Diego, CA). BCL Convert Conversion Software v3.9.3 (Illumina) demultiplexed FASTQ files. RNA-seq was performed by the University of Michigan