GSM4084059: ACL.ESCs.r2; Mus musculus; RNA-Seq
The isolated genomic DNA were used to construct the WGBS library, as previously described (Zhang et al., 2017). RNA-seq were performed under the instruction of mRNA library construction kit (NEB). Total RNA were extracted from approximately one million to two million cells using RNeasy Mini Kit (QIAGEN) according to the recommendation of manufacturer and then NEBNext® Poly(A) mRNA Magnetic Isolation Module was used to isolate mRNA from total RNA. Using mRNA as input, the first and second strand cDNAs were synthesized by NEBNext® RNA First Strand Synthesis Module and NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module, respectively. Final libraries were prepared using KAPA Hyper Prep Kits (8 PCR cycles) and sequenced on HiSeq4000 platform. Following the manufacturer's instructions, genomic DNA was extracted from stem cells using the DNeasy Blood & Tissue Kit (Qiagen). Remaining RNA was removed by treating with RNase A. Three replicated samples from each of these stem cells were used for library preparation to ensure repeatability of experiment. In short, 2 μg of genomic DNA spiked with 10 ng of lambda DNA were fragmented to about 300 bp with Covaris S220. Next, end repair and A-ligation were performed to the DNA fragments. Methylated Adaptor (NEB) was then ligated to the DNA fragments. In order to reach >99% bisulfite conversion, the adaptor-ligated DNA was treated twice using EZ-96 DNA Methylation-Direct™ MagPrep (Zymo Research). The resulting single-strand DNA fragments were amplified by 4 PCR cycles using the KAPA HiFi HotStart Uracil+ ReadyMix (2×). At last, the libraries were sequenced on HiSeq4000 platform to generate 150-bp paired-end reads.