title: GSM8289244: Uninfected, Rep 1, poly(A) depleted, rRNA depleted, mock digested; Homo sapiens; RNA-Seq
library_construction_protocol: Jurkat, 8B10, and 5F9 cells were lysed with TriZol (Thermo Fisher) and total RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo Research). The poly(A)-enriched RNA fraction was then isolated using the NEBNext® Poly(A) mRNA Magnetic Isolation Module, and the remaining poly(A)-depleted fraction was further processed using the NEBNext® rRNA Depletion Kit. The resulting poly(A)-depleted and rRNA-depleted RNA was treated with RNase R (or mock-treated with water instead of enzyme) under the following conditions: 1 unit RNase R enzyme (Lucigen) per microgram input RNA in a 20-50 µL reaction volume supplemented with 10X potassium chloride RNase R reaction buffer (Lucigen) incubated at 37°C for 2 hours. RNA was then purified from the digestion reaction using RNAClean XP magnetic beads (Beckman Coulter) and yields were quantified by Qubit. 30 ng of RNA was used for preparation of each library using the NEBNext® Ultra™ II Directional RNA Library Prep Kit, following manufacturer's instructions. Libraries were indexed using the NEBNext Universal PCR Primer for Illumina and unique indices selected from the NEBNext Multiplex Oligos for Illumina (Index Primer Set 1 and 2). Libraries were pooled and subjected to bulk RNA-sequencing on a NextSeq 500 platform using single-end 150 nt long reads, with a targeted depth of 15-20 million reads per poly(A)-enriched library and 50-60 million reads per rRNA-depleted library. The pooled library was sequenced twice to acheive the desired depth.