Abi prism 7500 sequence detection system

ChIP assay was performed using ChIP assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's protocol. In short, chromatin was sonicated 2 × 10 min to an average size of 300 - 800 bp. Prior to IP, 1 % of input was saved. Brg1, IGF-1R or IgG control antibodies were used for IP. Quantification of co-precipitated DNA was performed by qPCR using ABI PRISM 7500 Sequence Detection System, EpiTect Chip qPCR Primer (catalog number: GPH025770(−)01A) and RT² SYBR Green Master Mix (Qiagen, Hilden, Germany). The queried site primer detects the SNAI2 promoter (Human SNAI2, NM_003068.3 (−)01 kb). The cycling parameters were as followed: one cycle at 95°C for 10 min; 40 cycles of 95°C for 15 s and 60°C for 1 min.

The one-step RT-PCR kit (TransGen Biotech Co., Ltd., China) was used to assess the reverse transcription of total RNA. The primers used for polymerase chain reaction were as follows: α-SMA, forward—5′-AAGTATCCGATAGAACAC-3′, reverse—5′-AAACATAATCTGGGTCAT-3′; Runx2, forward—5′-AGGCGCATTTCAGATGATGAC-3′, reverse—5′-ACCTGCCTGGCTCTTCTTAC-3′; β-actin, forward—5′-GATGGTGGGTATGGGTCAGAAGGAC-3′, reverse—5′-GCTCATTGCCGATAGTGATGACT-3′. The ABI PRISM 7500 Sequence Detection System using QuantiTect SYBR Green (QIAGEN, Hilden, Germany) was used to quantify differential gene expression. The mRNA expression levels were normalized to β-actin mRNA.

We destructively sampled 10 mosquitoes per cage across all temperatures on days 8, 12, 16, and 20 post-Wolbachia infection. Mosquitoes were killed and immediately frozen at −80°C for future qPCR analyses. DNA was extracted from whole mosquito carcasses using the insect supplement in the 96-well DNeasy Blood and Tissue kit (Qiagen, Inc.) as per the manufacturer's protocol. We performed all qPCR analyses on an ABI Prism 7500 Sequence Detection System (TaqMan) using the Rotor-Gene SYBR Green PCR kit as per the manufacturer's protocol (Qiagen, Inc.) and the following cycler conditions (40 cycles total: activation, 95°C for 5 min; denaturation, 40 cycles run at 95°C for 5 sec each; and annealing and extension, 60°C for 30 sec). wAlbB was amplified from mosquito carcasses with modified GF and BR primers which specifically bind to the wsp gene54 . The relative abundance of wAlbB strain was determined after normalization to the mosquito single-copy rpS7 gene21 32 . All qPCRs were completed in triplicate, and the average efficiency of each assay was determined by quantifying the slope of a standard curve of five 1:10 serial dilutions from a positive sample in duplicate. Wolbachia densities (presented as a fold-change relative to rpS7 DNA) were generated using the qGENE software55 (link).