Cdna synthesis supermix

For verifying the results of RNA sequencing, qPCR assay was performed. Total RNA was extracted from the U2OS cells with and without TB treatment using RNAiso Plus (Takara Biotechnology Co., Ltd.), followed by cDNA synthesis using an All-in-One cDNA Synthesis SuperMix. Subsequently, the 2X SYBR-Green qPCR Master Mix kit was used for transcript quantification with specific primers. All the reactions were set up according to the manufacturers instructions. At the end of each reaction, a melting curve analysis was performed. Data were normalized to the expression of ACTIN and presented using the 2^−Δ∆Cq method (Table I) (19 (link)).

qRT-PCR was conducted as described previously.18 (link) The whole brain or PAs were homogenized and mixed with Trizol reagent for RNA extraction. The yields of RNA were assessed using a Nanodrop 2000 spectrophotometer. cDNA synthesis was conducted using a cDNA Synthesis Supermix (TAKARA, Japan). qRT-PCR assays were performed using the SYBR qPCR Supermix Plus (TAKARA, Japan). β-actin was used to normalize gene expression data. To detect miRNA expression, total RNA was reverse transcribed and then mixed with TaqMan Universal PCR Master Mix (TAKARA, Japan) and miRNA-specific TaqMan primers (Springen, Nanjing, China). MiRNA expression data were normalized to U6 RNA. The fold-change of gene expression was measured using the 2^−ΔΔCt method.18 (link) All primer sequences for qRT-PCR are presented in Supplementary Table S1.

By reference to the guidance of the manufacturer (Ambion, TX), cells were segregated by means of a PARIS kit. Concisely, OC cells (1×10⁷) underwent lysis in 1 mL cell segregation buffer and 15 min of centrifugation at 500 g. Next, TRIzol LS and TRIzol reagent (Invitrogen, USA) were independently employed to harvest the RNAs in the nuclear pellet and cell supernatant, and the total RNA was subjected to synthesis with the use of One Step gDNA Removal kit and cDNA Synthesis SuperMix (TaKaRa, China). QRT-PCR was then implemented thrice on a LightCycler 480 system (Roche, Switzerland) by means of an SYBR Premix Ex Taq kit (TaKaRa, China). QRT-PCR primer sequences are shown below: LINC00963, F, 5′-GGTAAATCGAGGCCCAGAGAT-3′, R, 5′-ACGTGGATGACAGCGTGTGA-3′; CHI3L1, F, 5′-GTGAAGGCGTCTCAAACAGG-3′, R, 5′-GAAGCGGTCAAGGGCATCT-3′; miR-378g, F, 5′-ACACTCCAGCTGGGGAAGACTGAGGTTC-3′, reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGCCCAGT-3′; GAPDH, F, 5′-CATGAGAAGTATGACAACAGCCT-3′, R, 5′-AGTCCTTCCACGATACCAAAGT-3′. 2^−ΔΔCt method was adopted for the calculation of mRNA and miRNA expression levels, which normalized to GAPDH or U6 level.