GSM7780247
Total RNA was isolated from cell lysates using the RNeasy Mini Kit (Qiagen) per the “Purification of Total RNA from Animal Cells using Spin Technology” section of the kit manual. An optional DNase I treatment was included and performed using the RNase-Free DNase Set (Qiagen) Bulk libraries were prepared using the NEBNext Ultra ii Directional RNA Library Prep for Illumina Kit (New England BioLabs) per Section 1 of the manufacturer's protocol. 500ng of total RNA was used as input for each sample, and an rRNA depletion was performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs). SPRIselect beads (Beckman Coulter) were used in place of NEB Sample Purification Beads for all purification steps. For the final enrichment PCR, bulk libraries were barcoded using the NEBNext Multiplex Oligos for Illumina – 96 Index Primers Kit (New England BioLabs) and amplified for 10 PCR cycles.