DNA Seq of TLR4 gene in Raw264.7 cells
Genomic DNA was evaluated with fluorometry by DeNovix QFX Fluorometer (DeNovix Inc., Wilmington, DE, USA; S/N: S-04083) using the Qubit dsDNA HS Assay Kit (Life Technologies, Waltham, MA, USA), and Nanodrop ND-8000 UV/Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA was used to set up PCR amplification. The primers used (TLR4-Forward-5-AGGAAGTTTCTCTGGACTAACAAG-3and TLR4-Reverse-5- ACTCATCAATGGTCACATCACA-3) were specific to flank the cut site. The PCR products were evaluated with agarose gel electrophoresis (Thermo Fisher Scientific, Waltham, MA, USA). PCR reaction products were cleaned up with AMPure XP beads and were evaluated with Fluorometry by DeNovix QFX Fluorometer. An indexed sequencing library was prepared from purified PCR products using the Twist Library Preparation Kit, Mechanical Fragmentation. The libraries were evaluated with Fluorometry by DeNovix QFX Fluorometer and with 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sample libraries were pooled with additional high-complexity sample libraries from other projects to generate the final run library. PhiX174 control library was added to the final run library at 5% (by content) of total DNA. Sequencing was performed on an Illumina NextSeq 1000 platform