title: GSM8673926: whole-cell RNA, endoplasmic reticulum, Nmd3 siRNA, biol rep 3 [ALIBi_Nmd3_Gnl3_KD_N3L]; Mus musculus; RNA-Seq
library_construction_protocol: Prior to affinity purification, an aliquot of cell lysate was reserved and mixed at a volume ratio of 1:9 with TRIzol (Thermo 15596-018) in an RNA non-stick tube. After affinity purification, eluate was mixed at a volume of 1:3 with TRIzol LS (Thermo 10296010) in an RNA non-stick tube. RNA was extracted using the DirectZol kit (R2060), following the manufacturer protocol without the on-column DNAse treatment. Extracted RNA was treated with Turbo DNAse (AM2239) per the manufacturer protocol and re-purified with an RNA Clean & Concentrator-5 column (R1016) per the manufacturer protocol. RNA concentration was estimated by Nanodrop 2000 (Thermo), and RNA integrity number (RIN) was assessed on an Agilent 2100 Bioanalyzer (total RNA pico) at the Stanford Protein and Nucleic Acid Facility. For each sample, 750 ng of RNA was used as input for the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs E7760S) with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs E7490) and indices from the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1 and 2) (New England Biolabs E7335S, E7500S), per the manufacturer protocol with 9 cycles of PCR amplification. Libraries were assessed on an Agilent 2100 Bioanalyzer (DNA 1000) to have an amplicon size of ~300 bp. Libraries were sequenced by Novogene on a Novaseq X or X Plus with paired-end 150 bp reads to a total target depth of 20 - 26 million read pairs per sample.