Rna mini kit

Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. RNA was quantified using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA). The RNA quality was determined using RNA 6000 Nano Kit (Agilent Technologies), all RNA samples had a RNA integrity number (RIN score) more than 7. Total RNA without any further DNase treatment was used for RNA sequencing. The ratio of human to mouse RNA in the xenograft samples was determined by measuring 18S and both mouse and human β2-microglobulin, hypoxanthine phosphoribosyltransferase 1 and actin beta expression with RT-qPCR. For subsequent cDNA synthesis TRIzol extracted RNA was cleaned up using a RNeasy Mini cleanup kit (Qiagen). The protocol also consisted of a DNase digestion step (Kit from Qiagen).
For FACS experiments, cells were directly sorted into lysis buffer. RNA was isolated using a RNA mini kit (Qiagen). Subsequently, RNA samples were amplified using a REPLI-g Single Cell RNA Library Kit.
For laser-captured micro-dissection analysis, RNA was isolated from specimens using a RNA mini kit (Qiagen).

Total RNA was isolated from MDMs transfected with MBOAT7 siRNA or control with and without LPS using the RNA mini kit (Qiagen). RNA purity and integrity were confirmed using an Agilent Bioanalyzer. Sequencing libraries were prepared from 100–500 ng of total RNA using the TrueSeq RNA sample preparation kit v2 (Illumina). Briefly, mRNA was purified, fragmented, and used for first- and second-strand cDNA synthesis followed by adenylation of 3′-ends. Samples were ligated to unique adaptors and subjected to PCR amplification. Libraries were validated using the 2100 BioAnalyzer (Agilent), normalized and pooled for sequencing. RNA-seq libraries prepared from three biological replicates for each condition were sequenced on the Illumina HiSeq 2000 using barcoded multiplexing and a 100 bp read length. Five biological replicates were performed.