Bioanalyzer rna pico kit

Total RNA was isolated from HEK293 and HeLa cells via Phenol/Chloroform extraction according to manufacturer’s protocol (Trizol, Thermo Fisher Scientific, MA, US). The BioAnalyzer System was used to measure the quality and quantity of total RNA. (BioAnalyzer RNA Pico Kit, Agilent, CA, USA). 2 μg of total RNA was used for RNA purification with magnetic bead cleanup module (Life Technologies, CA, USA). Purified RNA was ligated with sequencing adapters, reverse transcribed and purified (Ion Total RNA-Seq Kit v2, Life Technologies, CA, USA). Finally, barcodes were added and library size and amount was detected via BioAnalyzer HS Chip (Agilent CA, USA).

Fresh-frozen tissue samples were partially thawed in 1% formaldehyde/PBS (Phosphate-buffered saline) and continued to fix for 10 min at room temperature. Fixed tissue was rinsed in PBS and embedded in OCT (optimal cutting temperature) compound (4583 Tissue-Tek, Sakura Finetek, Tokyo, Japan). Tissue samples were sectioned (10 μm) and placed on membrane slides (Pen-membrane, Leica, Tokyo Japan). Sections were stained with hematoxylin and dehydrated by ethanol. Tissue parts were obtained by LMD (LMD6000, Leica) [12 (link)]. For the isolation of total RNA, sections were processed with an RNAeasy FFPE Kit (73504, Qiagen, Hilden, Germany). RNA yield and degradation status were monitored by a Bioanalyzer RNA Pico Kit (5067–1513, Agilent Technologies, Santa Clara, CA, USA). Total RNA samples (quantities in ng; Normal: range = 10.5–64.7, mean = 33.6; Tumor: range = 2.2–98.2, mean = 42.7; Stroma: range = 4.2–110, mean = 28.5) were converted into sequencing libraries using a SMARTer Stranded Total RNA Sample Prep Kit (635005, Takara Bio, Shiga, Japan). Sequencing was performed with Illumina NextSeq500 (Illumina, San Diego, CA, USA) to obtain 2 × 36-base reads.

For work on purified CD8⁺ T cells, freshly processed cells from the hospital cohort were sorted into 70% TRIzol LS Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −80°C. Both RNA and DNA were isolated from these samples using the TRIzol/chloroform method with minor modifications. Briefly, RNA was precipitated using a nucleic acid co-precipitant, 5 mg/mL linear acrylamide (Thermo Fisher Scientific). All reagents were kept at 4°C to facilitate separation of nucleic acid into different layers and efficient precipitation. QIAGEN RNeasy Micro kit was used to purify RNA from the TRIzol extracted samples. The quality of the extracted RNA was determined using the BioAnalyzer RNA Pico kit (Agilent, Santa Clara, CA, USA).

Stage 0 iNKT cells (PBS57–CD1d tetramer⁺, CD44⁻, CD24⁺, CD69⁺, CD4^lo/−, CD8⁻) were sorted from the pooled thymi of three BALB/c or SKG mice (~1 × 10³ cells were recovered) directly into Trizol buffer. Three independents samples were generated for each genotype. RNA quality was evaluated with the Bioanalyzer RNA pico kit (Agilent Technologies). Libraries were prepared from polyA⁺ RNA using the Whole Transcriptome Amplification Sequencing Technology SEQR kit (Sigma-Aldrich). Briefly, purified RNA was reverse transcribed using SuperScript II, Oligo dT30 VN primers, and template switching primers. A preamplification step of eight PCR cycles was performed using the Kapa HiFi Hotstart kit (Kapa Biosystems). The PCR product was purified using AMPureXP beads (Beckman Coulter) and 1 ng was further used for library preparation using the Nextera XT LibraryPrep kit (Illumina). Tagmented DNA was amplified with a 12-cycle PCR and again purified with AMPureXP beads. Library size distribution and yield were evaluated using the Bioanalyzer high-sensitivity DNA kit. Libraries were pooled at equimolar ratios and sequenced with the rapid run protocol on a HiSeq. 2500 (Illumina) with 50-nt single end cycling.