Methyl primer express software

Based on the use of Methyl Primer Express software (Applied Biosystems, Foster City, CA, USA), we carried out a search for the possible presence of CpG island within a range of 1000-nt of transcription start site upstream of goat CDK19 gene in goat genome (assembly ARS1, NC_030816.1: 26801290-26972150, https://www.ncbi.nlm.nih.gov/genome/?term=goat, accessed on 28 July 2019). We predicted the potential binding sites of transcription factors within the BSP amplification region (544-nt) by the AliBaba 2.1 program (http://gene-regulation.com/pub/programs.html). From SHF stem cells, the genomic DNA was isolated and treated with MethylCode Bisulfite Conversion Kit (Invitrogen, Shanghai, China). Within the revealed CpG island (680-nt), we designed a pair of primers (BSP-F and BSP-R). A total of 24 CpG sites were included within their potential amplification region. Bisulfite sequencing PCR reactions were performed, and the amplified products were subjected to purification and were cloned into competent E. coli DH5α cells. We sequenced 10 positive clones in each group of cells, and the results were displayed using the QUMA program [21 (link)].

For the validation of microarray discovery we performed Methylation-Sensitive High Resolution Melting (MS-HRM) analysis on a group of genes showing differential methylation in the HIV seropositive (HIV+)/METH+ group, including NET1; TTL7; TGFBR3; SCN1A; UNC5D and APBA1. Primers were designed for bisulfite-converted DNA using Methyl Primer Express software (Applied Biosystems) to produce an amplicon <300 bp that overlapped the Illumina probe-set that showed differential methylation and also included neighbor CpG sites to increase the magnitude of the delta of their melting temperatures (Tm). Presence of CpGs in the primer sequence was avoided to prevent bias due to preferential amplification of the unmethylated target DNA [34] . Primer efficiency and specificity was tested by running a mock MS-HRM using 0% and 100% methylated human standard DNA (Life Technologies) and analyzing the profile of Tm peaks in the melting curve and the number and size of amplicons by gel electrophoresis. Bisulfite-converted DNA templates (40 ng) from the studied cases underwent PCR alongside with standards ranging from 0% to 100% methylation using MeltDoctor HRM Dye (Life Technologies). Melt curves of the samples were fitted to the sample curves using HRM 3.0 software (Applied Biosystems).

To determine the DNA methylation levels in the MITE excision lines and the wild types, the genomic DNA was isolated from leaves at 60-day after germination using the DNeasy Plant Mini Kit (QIAGEN). Bisulfite treatment was performed with the EpiTect Bisulfite kit (Qiagen) following the manufacturer's instructions. The treated DNA was used for PCR amplification with primers at the Ghd2 locus. The primers were designed by Methyl Primer Express software (Applied Biosystems). Primers for bisulfite sequencing were listed in the Supplementary Table 2. All PCR fragments were cloned to pGEM-T easy vector (Promega) and 20 clones for each fragment were sequenced. The sequencing data were analysed on the web-based kismeth bisulfite analysis sofeware69 (link).

For bisulphite conversion, 800 nanogram of DNA was bisulphite converted using the EZ DNA Methylation Kit (Zymo Research, Orange, CA). PCR was carried out using primers (see Supplementary Table S1 for the primer sequences) designed by Methyl primer express software (Applied Bio systems). 1 μl (10–30 ng) of bisulphite converted DNA was PCR amplified under following cycling conditions: 95 °C for 3 min, 40 cycles of 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 2 min, followed by one time delay cycle of 72 °C for 5 min and a 4 °C soak. Amplification of the target region was verified by gel electrophoresis on a 2% agarose gel. Samples were purified using the QiagenQiaquick gel extraction kit, quantified through Nanodrop spectrophotometer (ND-1000, Thermo Scientific) and sequenced using forward or reverse primers. Sequencing was done according to the previously reported method20 . Results were analyzed using BIQ analyzer software21 (link). These studies were performed in three replicates from the samples isolated from three different rat brains.