For bulk RNA sequencing in a small number of cells (50 to 500), we collected the cells using a robot, briefly spun them down at 500×g, and then performed reverse transcription and pre-amplification using the Smart-seq2 protocol [51 (link)]. All primers (Genewiz) used for reverse transcription and pre-amplification were biotinylated. We performed post-PCR cleaning with Ampure XP Beads (Beckman Coulter, A63881) at a ratio of 0.8/1 (beads/cDNA). To quantify the cDNA, we used PicoGreen (Yeasen, 12641) with FlexStation3 (Molecular Devices) and assessed the quality busing Qsep100 with S2-Standard Cartridge Kit (BiOptic). Following this, sequencing libraries were generated using the TruePrep Flexible DNA Library Prep Kit for Illumina (Vazyme, TD504) and sequenced them using the Illumina NovaSeq 6000 sequencer.
Picogreen
Manufactured by Yeasen
Sourced in China
PicoGreen is a fluorescent nucleic acid stain used for quantifying double-stranded DNA (dsDNA) in solution. It provides a sensitive and accurate method for measuring dsDNA concentrations in samples.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Mitotracker, by Beyotime (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Fetal bovine serum (fbs), by Biosharp (1 mentions)
Lentivirus expressing luciferase, by Hanbio Biotechnology (1 mentions)
Ncrc 10001 50, by Cyagen (1 mentions)
Actin tracker green 488, by Beyotime (1 mentions)
Rpmi 1640 medium, by Biosharp (1 mentions)
Penicillin streptomycin, by Biosharp (1 mentions)
Ab80826, by Abcam (1 mentions)
Ab240087, by Abcam (1 mentions)
4 protocols using picogreen
1
Bulk RNA-seq from Small Cell Samples
2
Mitochondrial DNA Extravasation Assay
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The cells were divided into control and MT and DHT double-deficient groups. WPMY-1 cells were seeded into confocal dishes at 5 × 103 cells. The discarded supernatant was washed with serum-free medium three times, and serum-free medium containing PicoGreen (Yeasen, Shanghai, China) and MitoTracker (Beyotime, Shanghai, China) was added and incubated at 37 °C for 30 min. The cells were washed 3 times in serum-free medium. Laser scanning confocal microscopy (LSM800, Zeiss) was used to monitor mitochondrial DNA extravasation into the cytoplasm.
3
Cell Line Engineering and In Vivo Assays
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Lipofectamine 3000 was bought from Thermo Fisher Scientific (L3000015). The cryopreservation medium (NCRC-10001-50) was purchased from Cyagen Biosciences. CDK4 CRISPR-Cas9 knockout plasmid (sc-400148) was purchased from Santa Cruz. Actin-Tracker Green-488 (C2201S) was bought from Beyotime Biotechnology. Lentivirus expressing luciferase was purchased from HANBIO. A TUNEL assay kit (Red AF647) was obtained from Procell. Picogreen was purchased from Yeasen Biotechnology. A549 and H226 cell lines were obtained from the American Type Culture Collection. A549 and H226 cell lines were cultured in F12K medium and RPMI 1640 medium, which contain 10% fetal bovine serum and penicillin/streptomycin (100 U ml−1; Biosharp) at 37°C in 5% CO2. Five-week BALB/c nude mice (female/male) were obtained from Zhejiang Vital River Laboratory Animal Technology. Mouse study was conducted in accordance with the protocol approved by Animal Ethics Committee of Zhejiang University (ZJU20230329).
4
Immunofluorescence Assay for Cellular Responses
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After treatment with drugs for 72 hours, 3 µg/mL PicoGreen (12641ES01, Yeasen) was added to the medium. After 1 hour, cells were fixed (4% paraformaldehyde), permeabilized (0.25% TritonX‐100) and blocked (5% BSA). Cells were then stained with anti–pan Cytokeratin (ab80826, Abcam), phospho‐Histone H2A.X (9718, CST), phosphor‐TBK1 (5483, CST), phospho‐IRF‐3 (29047, CST), and anti–MHC‐I (ab240087, Abcam) (ab281902, Abcam) overnight at 4°C. Cells were stained with secondary antibody for 1 hour and then counterstained with DAPI. Photographs were taken using confocal microscopy.
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