Nanodrop nd 1000 spectrophotometer

Total RNA was obtained from cultured cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. The concentration of isolated total RNA was measured with a NanoDrop ND‐1000 Spectrophotometer (Agilent, CA, USA). Genomic DNA was eliminated and RT was performed using the PrimeScript RT Reagent kit with gDNA Eraser; qPCR was performed using the SYBR Green PCR kit (both Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's recommendations. The process was conducted using a 7900 Real‐Time PCR System (Applied Biosystems; Thermo Fisher Scientific Inc.), β‐actin was used as an internal reference. The primer sequences are shown in Table 1.

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany). The concentration of isolated total RNA was measured by NanoDrop ND-1000 Spectrophotometer (Agilent, CA). The qRT-PCR was performed using SsoFast™ EvaGreen® Supermix (Bio-Rad) with hypoxanthine phosphoribosyltransferase 1 (HPRT1) as an internal control, as described previously [18 (link)] and Supplementary Materials and Methods.

The total RNA of the ‘leaf’ and ‘pet’ samples was isolated and purified using Trizol reagent (Invitrogen, CA, USA). The content and quality of the RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (CA, USA).
To analyze the expression of mRNAs, lncRNAs, and circRNAs, a Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA) was used to remove ribosomal RNA for construction of the chain-specific library. Each of the ‘pet’ and ‘leaf’ samples comprised three biological replicates, and hence a total of six libraries (‘leaf 1,’ ‘leaf 2,’ ‘leaf 3,’ ‘pet 1,’ ‘pet 2,’ and ‘pet 3’) were prepared. The qualified libraries were sequenced using the Illumina NovaSeq™ 6000 system (LC-BIO, China). The read length of the double-ended sequence was 2 × 150 bp.
A total of six small RNA libraries was prepared using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA) to analyze miRNA expression. The prepared libraries were sequenced using the Illumina HiSeq 2000/2500 system (LC-BIO, China). The single sequence read length was 1 × 50 bp.

Total RNA from cells and tissues was isolated using Trizol extractions (Invitrogen). The RNA quantity was assessed by NanoDrop®ND-1000 spectrophotometer (Agilent, Palo Alto, USA). 100 ng of total RNA was amplified using the Ambion® WT Expression Kit (4411973, Life Technologies). Then 5.5 μg of the cDNA was fragmented and labeled with the GeneChip® WT Terminal Labeling kit (901525, Affymetrix). Libraries were sequenced either on Illumina HiSeq 2000 or HiSeq 2500 using v3 chemistry.
Followed by background deletion, quantile normalization, and probe assembly. Different expression genes (DEGs) between normal vs. tumor tissues were detected by the empirical Bayes method [27 (link)]. The p-values were adjusted for multiple comparisons using the Benjamini-Hochberg procedure [28 (link)]. Genes with adjusted p-value < 0.05 and |log FC| ≥ 1.0 were considered as differentially expressed. Enrichment analysis of DEGs was performed with DAVID [29 (link)] and ClueGO [30 (link)]. The enriched GO (BP: biological process; CC: cellular component; MF: molecular function) and pathway terms were listed with participant genes [31 (link)]. Some other databases used are listed in Table 2.

Total RNA was prepared from K562 mbIL-21 expanded control and TGFβi NK cells as per manufacturer’s instructions using the Total RNA Purification Plus Kit (Norgen Biotek, Thorold, ON, Canada) and the resulting total RNA was quantified in a Nanodrop ND-1000 spectrophotometer, and checked for purity and integrity in a Bioanalyzer-2100 device (Agilent Technologies Inc., Santa Clara, CA, USA) and submitted to the genomics core at the Nationwide Children’s Hospital for sequencing. Libraries were prepared using the TruSeq RNA Sample Preparation Kit (Illumina Inc., San Diego, CA, USA) according to the protocols recommended by the manufacturer. The quality of the libraries were determined via Agilent 4200 Tapestation using a High Sensitivity D1000 ScreenTape Assay kit and quantified by KAPA qPCR (KAPA BioSystems, Cape Town, South Africa). Approximately 60–80 million paired-end 150 bp sequence reads per library were generated using the Illumina HiSeq4000 platform.