Qubit rna hs assay kit

RNA was extracted with miRNeasy micro kit from Qiagen, following manufacturer's instructions with minor modifications. Briefly, Qiazol extracts were thawed and vortexed for 1min. Chloroform was applied and mixture was transferred to a MaxTract High density column. From that point on, the extraction was then performed according to the kit's manual for low input samples. RNA was then measured with QuBit RNA HS assay kit and Agilent RNA 6000 Pico Kit. 50 ng of RNA from each E13.5/ P7 sample was used for library preparation. Each sample represented a pool of Pdgfra+ cells collected from 4 – 32 animals to achieve the 50ng required for library preparation.

The A549 cells under normal conditions (n = 3, blank group) and hypoxic A549 cells (n = 3, hypoxic group) were collected and sent to Yanzai Biotechnology (Shanghai) Co. Ltd. (Shanghai, China) for whole-transcriptome sequencing, including lncRNA and miRNA sequencing. Briefly, total RNA was isolated from each sample using the RNAiso Plus kit (Trizol, Takara Biomedical Technology Co., Ltd.) following the manufacturer’s recommendations. Then, the purity, concentration, and integrity of the total RNA were evaluated by NanoDrop™ One/OneC, Qubit™ RNA HS Assay Kit, and Agilent 4200 TapeStation system, respectively. After library construction of the lncRNAs and miRNAs, each sample was sequenced on an Illumina platform.
After data preprocessing and analysis, clean data were mapped to their corresponding databases using HISAT2 and Burrows-Wheeler Aligner software. Subsequently, differentially expressed miRNAs (DEmiRNAs) and differentially expressed lncRNAs (DElncRNAs) between normal A549 cells and hypoxic A549 cells were screened using the DESeq algorithm in the R package with the criteria of |log₂Fold change (FC)| > 1 and adjusted p-value (padj) < 0.05. The identified DE miRNAs and DElncRNAs were subjected to functional analyses, including Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Statistical significance was set at P < 0.05.

For sample extraction, 140 µL of cell culture supernatant was used with the QIAamp^® Viral RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s recommendations. Total RNA was quantified using the Qubit^® 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) with the Qubit^® RNA HS Assay kit and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) with the Agilent RNA 6000 Pico kit following the respective manufacturer’s protocols. Subsequently, the samples were stored in a freezer at −70 °C until further use.

The fluorescence-activated cell sorting (FACS) protocol was adapted from (Harzer et al., 2013 (link)). Briefly, approximately 50 UAS-Lifeactin.GFP; sr-gal4/TM6b, tb white pupae (0 h APF) were dissected on ice to collect 250–300 leg imaginal discs in M3 (S3652 Sigma Aldrich) complemented medium. Leg disc cells were dissociated in collagenase (P4762-Sigma Aldrich) and papain (C267-Sigma Aldrich) solution for 1 h at 30°C, 300 rpm (Thermomixer Eppendorf), with additional mechanical stirring. After filtering, we used a FACSAriaTM device (4 °C, 20 psi, nozzle ∅ 100 μm) to first sort a batch of cells (roughly 50,000 cells per sample) independently of fluorescence, which we used as input (IP) and we then sorted the tendon cell (GFP+) population based on GFP fluorescence. Samples were directly collected in Trizol reagent (Invitrogen, 15596026). RNA extraction was performed with Zymo Quick-RNA microprep kit (R1051), sample RNA quality and quantity were estimated with QuBit (ThermoFisher, Qubit RNA HS Assay Kit, Q32852) and BioAnalyzer (Agilent, Agilent RNA 6000 Nano Kit 5067-1511), and sample specificity was analyzed by qPCR. Samples were stored at−80°C. For the detailed protocol, see Supplementary Material S1.