| Library |
| Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
|
For tissues, we collected fetal tissues from four healthy pregnant Holstein cows at a slaughterhouse. After the slaughter, we extracted fetal tissues to examine their normal development and measured the crown-rump length (CRL) to estimate gestational age. The gender was identified based on the position of the gonads within the abdominal cavity, anatomical structures, and relationships with adjacent organs (mesonephros and/or kidneys). Two male and two female fetuses were identified (13-16 cm CRL), nine different tissues were collected, including the forebrain, hindbrain, heart, liver, lungs, skeletal muscle (hind limb), kidneys, rumen, and testes. Genomic DNA degradation and contamination was monitored on agarose gels. DNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). DNA concentration was measured using Qubit DNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA). A total amount of 5.2 microgram genomic DNA spiked with 26 ng lambda DNA were fragmented by sonication to 200-300bp with Covaris S220, followed by end repair and adenylation. Cytosine-methylated barcodes were ligated to sonicated DNA as per manufacturer’s instructions. Then these DNA fragments were treated twice with bisulfite using EZ DNA Methylation-GoldTM Kit (Zymo Research), before the resulting single-strand DNA fragments were PCR amplificated using KAPA HiFi HotStart Uracil + ReadyMix (2X). Library concentration was quantified by Qubit 2.0 Flurometer (Life Technologies, CA, USA) and quantitative PCR, and the insert size was assayed on Agilent Bioanalyzer 2100 system. |
Bisulfite-Seq |
GENOMIC |
PCR |
PAIRED
|
|
