Genepaint

We performed RNA in situ hybridization (ISH) experiment by using digoxigenin-labeled RNA probes, a non-radioactive technique based on dual signal amplification, named ‘CARD’. RNA ISH was carried out on highly automated robotic equipment (TECAN GenePaint). The minimum amount of RNA probe used was 25 ng/slide. Each lncRNA and mRNA gene was analyzed on 24 sagittal sections of mouse embryonic E14.5 and 20 sagittal sections of P56 brain tissue sections. The entire protocol was performed according to method in the study of Eichele et al. (45 (link)).

Several samples from different testes were sectioned (4 μm) and placed on a single slide; each had sections from different mice of the same group, with the addition of one WT control animal on each slide. The slides were deparaffinized (Histoclear; Diamed Inc.) and loaded in 30-slide batches into the Tecan GenePaint (Tecan) solvent delivery robotic system, programmed to rehydrate the tissues by passing them through a graded ethanol series, and then stained with Periodic Acid Schiff (PAS) (Sigma) according to the manufacturer's protocol. The slides were mounted with Permount (Fisher Scientific) and coverslips and left to dry before observation with a light microscope. Tubule diameters and epithelial heights were measured using ImageJ (NIH) for 200 tubules per animal, with five to six animals per experimental group. Using similar methods, a fixed epididymis from reach mouse was sectioned (4 μm) and stained using Toluidine Blue (Sigma) following the manufacturer's protocol.

Methylation analysis was performed using Illumina®MethylationEPIC Beadchips prepared as described in the Illumina® Infinium® HD Assay Methylation Protocol Guide (15019519 v01) before processing on the Illumina iScan System. Input DNA (250 ​ng) was bisulfite treated using the Zymo EZ DNA Methylation Kit. Zymo’s Human Methylated and Non-methylated DNA controls are treated with samples. Controls are PCR amplified and run on a gel to confirm both methylated and unmethylated bands are present. After bisulfite conversion was confirmed, the bisulfite treated DNA was manually prepared for sequence-specific array-based hybridization using whole-genome amplification (WGA), enzymatic endpoint fragmentation and chemical precipitation. The WGA product was re-suspended and captured by array hybridization. Arrays were then mounted in the Tecan GenePaint automated slide processor on the Tecan Freedom Evo® robotic liquid handling system for primer extension and staining. The amount of fluorescence was measured and used to determine the methylation level of the CpG sites.