Hiseq v4 single read flow cell

MDM were generated as described and stimulated with 25 ng/ml of the indicated IFNs. Total RNA was isolated 18 h following stimulation using RNeasy minikit (Qiagen). Intact poly(A) RNA was purified from total RNA samples (100 to 500 ng) with oligo(dT) magnetic beads, and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation kit (catalog no. RS-122-2101 and RS-122-2102). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (catalog no. 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant kit (catalog no. KK4824). Individual libraries were normalized to 10 nM, and equal volumes were pooled in preparation for Illumina sequence analysis. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot system. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR cluster kit v4-cBot (catalog no. GD-401-4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS kit v4 sequencing reagents (catalog no. FC-401-4002).

Total RNA isolated via miRNeasy Qiagen RNA isolation kit was submitted to the University of Utah genomics core facility, a cDNA library was prepared and Illumina HiSeq 2500 sequencing was performed as in^{67 (link)}, with modifications. Briefly, RNA was quantified with a Qubit RNA HS Assay Kit (Fisher Scientific #Q32855). RNA quality was evaluated with an Agilent Technologies RNA ScreenTape Assay (5067-5579 and 5067-5580). After rRNA depletion reactions on 100–500 ng total RNA, stranded libraries were prepared using the Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Gold (RS-122-2301 and RS-122-2302). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (5067-5582 and 5067-5583). Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis. Libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell, amplified and annealed to sequencing primers, and 50 cycle single read runs were performed using an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), and corresponding reagent kits (GD-401-4001, FC-401-4002). Resulting data were aligned to the mouse mm10 reference genome (mm10, M_musculus_Dec_2011, GRCm38).