Kapa stranded rna seq kit with riboerase hmr

Sixteen hours prior to the experiment, cells were seeded at a density of 7.5 × 10⁵ cells/mL in a 6-well plate. After overnight incubation, cells were treated with 2.5 μM EPH334 (1, 8, and 16 h) or 1 μM of SAHA (2, 4, and 8 h) in triplicate. Cell pellets were then collected and stored at −80 °C. Total RNA was isolated using the Quick-RNA Miniprep Kit (Zymo, Cat. No. R1055) according to manufacturer’s instructions. RNA quality was determined with Agilent Tapestation ScreenTape (Agilent, Santa Clara, CA, USA, Cat. No. 5067-5576). An amount of 1 µg of total RNA with ERCC Spike-ins (ThermoFisher, Waltham, MA, USA, Cat. No. 4456740) was used to prepare libraries with KAPA Stranded RNA-seq kit with RiboErase (HMR) (KAPA Biosystems, Wilmington, MA, USA, Cat. No. KK8483) according to manufacturer’s instructions. After final cleanup, libraries were run on Agilent Tapestation DNA D1000 ScreenTape (Agilent, Cat. No. 5067-5582) to confirm fragment size and quantified using a Qubit 2.0 fluorometer. Libraries were submitted to the UT Southwestern McDermott Center Sequencing Core and 75 bp paired-end (PE) reads were generated for both libraries.

We collected serial blood samples, post-treatment primary tumor samples, and draining lymph nodes specimens from four patients. Genomic DNA was extracted from frozen tumor and normal tissue sections using the QIAamp DNA FFPE Tissue Kit (Qiagen) following the manufacturer’s instructions. A minimum of 1ug of DNA was used for WES profiling experiment. The DNA quality was assessed by Nanodrop2000 (Thermo Fisher Scientific), and the quantity was measured by the dsDNA HS Assay Kit using Qubit 2.0 (Life Technologies). WES was only performed when the tumor proportion was above 20%. WES library was sequenced using an Illumina Novaseq 4000 platform according to the manufacturer’s instructions.
Total RNA from frozen section samples was extracted using RNeasy FFPE kit (QIAGEN). Ribosomal RNA was depleted using RNase H followed by library preparation using KAPA Stranded RNA -seq Kit with RiboErase (HMR) (KAPA Biosystems), and library quality was accessed by Agilent High Sensitivity DNA kit on Bioanalyzer 2100 (Agilent Technologies), which was then sequenced on Illumina Novaseq NGS platforms.

Total RNA from frozen samples was extracted using the RNeasy Mini Kit (QIAGEN). Ribosomal RNA was depleted using RNase H followed by library preparation using KAPA Stranded RNA‐seq Kit with RiboErase (HMR) (KAPA Biosystems). Library concentration was determined by KAPA Library Quantification Kit (KAPA Biosystems), and library quality was accessed by Agilent High Sensitivity DNA Kit on Bioanalyzer 2100 (Agilent Technologies), which was then sequenced on Illumina HiSeq NGS platforms (Illumina).

The FFPE tumor and paired normal samples of 45 patients were selected from 54 patients with LUAD for mRNA sequencing. The percentage of tumor cells in these samples should be more than 80%. Among the patients, 25 out of 45 were tested positive for SHOX2 promoter methylation and 18 out of 45 were tested positive for RASSF1A promoter methylation. Total RNA from samples was extracted using miRNeasy FFPE kit (QIAGEN). Ribosomal RNA was depleted using KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA Biosystems). Library preparations were performed with KAPA Stranded RNA-seq Library Preparation Kit (Roche). Library concentration was determined by KAPA Library Quantification Kit (KAPA Biosystems), and library quality was accessed by Agilent High Sensitivity DNA kit on Bioanalyzer 2100 (Agilent Technologies), which was then sequenced on Illumina HiSeq NGS platforms (Illumina). The amount of sequencing data for each sample was 30M.

Total RNA was isolated by using the QIAGEN AllPrep Kit. RNA sequencing libraries were prepared using the KAPA Stranded RNA-Seq Kit with RiboErase (HMR; KAPA Biosystems). RNA was fragmented to a size of 100–200 nucleotides and amplified for 11 cycles. Final libraries were quantified on a high-sensitivity bioanalyzer chip and sequenced on the NextSeq 550 (Illumina).