Dnase 1

Prostate cancer cells were lysed with RIPA buffer containing RNase inhibitor (CWBio, Beijing China) and cell lysates was incubated with biotin-labeling oligonucleotides for the specific-region sets of TIMP2 and TIMP3 overnight at 4°C. Then the biotin-oligonucleotides-protein-RNA complexes were pulled down with ImmunoPure streptavidin-agarose beads (20 μl/sample, Pierce, Rockford, IL, USA) and the complexes were freed from streptavidin-agarose beads by using 0.1 M biotin. The solution containing the complexed was treated with DNase I (CWBio) and Proteinase K (CWBio) before the RNA extracted with Trizol reagent, and then the RNA was reverse-transcribed to cDNA and PCR was performed to detect DANCR, and the primer sequences are as followings: F:5′-GCCACTATGTAGAGGGTTTC-3′, R:5′-AC-CTGCGCTAAGAACTGAGG-3′.

The nucleic acids and proteins in sCABs were obtained with TRIzol according to the manufacturer's instructions. After the enzymatic hydrolysis reaction with DNaseI (CW2090s, CWBIO, China) and RNaseA (EN0531, Thermo Scientific, USA) separately, the concentrations of RNA and DNA were analyzed based on the absorbance at OD₂₆₀ by the NanoDrop One instrument (Thermo Fisher Scientific, USA). The samples of RNA and DNA were then separated by urea‐denatured polyacrylamide gel electrophoresis (5%) and a 1% agarose gel. The gels were imaged with a UV transilluminator (Gel Doc 2000, Hercules, USA) after ethidium bromide (0.1%) staining.
The proteins were washed with guanidine hydrochloride (0.3 м) at least 2 times and resuspended in sodium dodecyl sulfate (SDS) (200 μL, 1%). The samples were then subjected to LC‐MS analysis on a Shimadzu UFLC 20ADXR HPLC system connected in line with an AB Sciex 5600 Triple TOF mass spectrometer (AB SCIEX, USA). The proteins were analyzed by searching the mouse taxon of the UniProtKB/SwissProt database (release 2011_11). Gene ontology (GO) (http://geneontology.org/) was used to classify the proteins based on cellular components and molecular functions. The proteins were clustered according to pathway terms identified from KEGG database.

Total RNA from antennae, proboscis and maxillary palps was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was quantified and qualified using a NanoDrop ND-1000 spectrophotometer and 2% agarose gel electrophoresis, respectively. The RNA was treated with DNase I (Cwbio, Beijing, China) to remove genomic DNA contamination. Approximately 1 μg of RNA was reverse transcribed into the first-strand cDNA using the SuperScript III RT Kit (Invitrogen, Carlsbad, CA, USA). The RT-qPCR was performed using PrimeScript™ RT Master Mix (Takara, Dalian, China) in a 25-μl system that contained 200 nM forward and reverse primers, 200 μM dNTPs, 2.5U TB Green Premix Ex Taq II and 1 μl cDNA template (approximately 40 ng). The thermal cycling conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Three biological and three technical replicates were performed for each sample. The data were analyzed by the 2^−ΔΔCT method [53 (link)], and results were expressed as log₂-transformed fold change values. A housekeeping gene, RpS7, was used as an internal control. Gene-specific primers that spanned exon-intron boundaries were designed using Primer 5.0 and are listed in Additional file 1: Table S1.