title: GSM8940730: pre-treatment tumor RNA from individual with NSCLC_S024; Homo sapiens; RNA-Seq
library_construction_protocol: Total nucleic acid (TNA) was extracted from tissue specimens using a Chemagic 360 or similar instrumentation, and the recovered TNA was quantified and qualified, and may have been concentrated if necessary. Extracted TNA that was intended for processing by Tempus|RS was treated with Invitrogen TURBO DNase to degrade DNA. The minimum amount of input total RNA required to perform the test was 50 ng. RNA was fragmented using heat and magnesium, with variable parameters, to yield similar sized fragments from RNA inputs with different starting size distributions. Strand-specific library preparation was performed using the KAPA RNA HyperPrep Kit for Illumina with KAPA single indexed 6 base pair adapters. The libraries were amplified with high fidelity, low-bias PCR using primers complementary to adapter sequences. After library preparation and amplification, targets were captured by hybridization, clean-up of captured targets was performed, and unbound fragments were washed away. Library capture is conducted using the xGen Exome Research Panel v1 probe set, along with the xGen Hybridization and Wash Kit and xGen Universal Blockers. The enriched targets are amplified using the KAPA HiFi HotStart ReadyMix and primers supplied in the KAPA Library amplification kit, followed by a magnetic bead-based clean-up. The amplified target-captured libraries were sequenced with a 2x76 read length to an average of 50 million total reads on an Illumina HiSeq4000 System using patterned flowcells.