Magmax 96 total rna isolation kit

RNA was extracted from blood and homogenized tissue samples using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific, https://www.thermofisher.com) on a 96-well ABI MagMAX extraction platform with a DNase-I treatment step, according to the manufacturer’s instructions. RNA was quantified by a quantitative reverse transcription PCR (qRT-PCR) targeting a strain-specific nucleoprotein gene sequence (primer and probe sequences available on request from the authors), and normalized to 18S RNA levels. We determined viral small (S) segment copy numbers using standards prepared from in vitro–transcribed S segment RNA.

RNA was isolated from all four fractions (TH, CD11b+, ACSA2+, CD11b/ACSA2-) using the MagMax-96 Total RNA Isolation Kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). The RNA yield was quantified on a NanoDrop 1000 spectrophotometer and assessed for quality on an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). RNA was reverse transcribed into cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc.). cDNA was tested for genomic DNA contamination and showed at least a 10 Cq difference between the +RT (reverse transcription) and –RT samples
^{38 (link)}. Applied Biosystems TaqMan® Gene Expression Assay (Thermo Fisher Scientific Inc.) primers were used, and specific assay IDs are shown in
Table S1. RT-qPCR reactions were performed using SsoAdvanced™ Universal Probes Supermix (BioRad, Hercules, CA, USA) in 10-μL reactions containing 250 pg of cDNA. All reactions were performed in technical triplicates for each biological replicate and included a negative no-template control. Samples were normalized to 18s rRNA and relative expression was determined using the CFX software version 3.1 (BioRad).

LSCC and LADC cells were isolated as explained above. RNA was isolated using the MagMAX-96 total RNA Isolation Kit, following the manufacturer’s instructions (Thermo Fisher Scientific). Read mapping and abundance estimation were performed using RSEM v1.2.31 (Li and Dewey, 2011 (link)) running STAR v2.5 (Dobin and Gingeras, 2015 (link)) against the Mus musculus Ensembl 86 transcriptome (GRCm38), with the following nondefault command line arguments: “–star–forward-prob 0.” Further analysis was conducted using R v3.4.0 running Bioconductor v3.5. Gene-level read count estimates were rounded to integer values and used by DESeq2 (Love et al., 2014 (link)) to assess differential expression between LSCC and LADC replicate groups using the “DESeq2” function with default settings.
Normalized counts were used to construct an expression heatmap for LSCC signature genes common to two previous studies (Xu et al., 2014 (link); Ferone et al., 2016 (link)). For the purposes of visualization, gene counts across samples were converted to z-scores and ordered by magnitude of change.
RNASeq data were deposited in the Gene Expression Omnibus (GEO) repository under accession no. GSE123716.

Selected BM CD34+/lin- cells of 80 CP-CML patients were resuspended in 50 μl of RNAlater (Thermo Fisher Scientific, Milano, Italy) and stored at -20°C until RNA extraction was performed as previously described [18 (link)].
Total RNA was isolated from the BM CD34+/lin- cells stored in RNAlater using MagMAX 96 Total RNA Isolation Kit (Thermo Fisher Scientific) [18 (link)], according to the manufacturer’s instructions. The quality and the yield of the extracted RNA were measured using Nanodrop (Thermo Fisher Scientific) (see http://dx.doi.org/10.17504/protocols.io.yncfvaw).