GSM5218936: bESC-IVF_rep3_IgG; Bos taurus; OTHER
3'-Tag-RNA-seq: RNA was isolated from fibroblasts and bESC devoid of MEF using the RNeasy Mini Kit (74104, Qiagen) including DNase treatment with the RNase-Free DNase Set (79254, Quiagen) following manufacturer's instructions. RNA was quantified using the Qubit RNA BR Assay Kit (Q10211, Invitrogen) and its integrity was studied by determining the RNA quality index (RQI) using a Bio-Rad Experion™ Automated Electrophoresis System. 3'-Tag-RNA-seq: Isolated RNA was submitted to DNA Technologies and Expression Analysis Core at the University of California Davis Genome Center for library preparation and sequencing. Sequencing was performed on the Illumina HiSeq 4000 platform and produced single-end 90 bp reads. CUT&Tag was performed as previously described (Kaya-Okur et al., 2019). The protocol was adapted for 100,000 cells per sample. Briefly, cells were thawed as previously described, then washed and incubated with conditioned ConA bead slurry (10 µl per 100,000 cells) for 10 min at room temperature in an end-over-end rotator. Cells were then incubated overnight on a nutator at 4°C with a 1:50 dilution with primary antibody. Afterwards, cells were incubated with the secondary antibody guinea pig anti-rabbit IgG antibody (ABIN101961, AntibodiesOnline) in a 1:100 dilution for 1 h at room temperature using a nutator. Cells were then washed and mixed with the pA-Tn5 adapter complex in a 1:250 dilution for 1 h at room temperature in a nutator. Following incubation with the pA-Tn5 adapter complex, cells were washed and then incubated in tagmentation buffer for 1 h at 37°C.  After 1 h, tagmentation was stopped and DNA fragments were solubilized by adding 10 μL 0.5M EDTA, 3 μL 10% SDS and 2.5 μL 20 mg/mL Proteinase K to each sample, followed by full-speed vortexing and incubation for 1 h at 50°C. DNA fragments were extracted using phenol-cloroform-isopropide procedure and any remaining RNA was degraded by incubating the samples in a 1:400 RNAse A dilution for 10 min at 37°C. Finally, DNA fragments were amplified with the universal i5 primer, barcoded i7 primers, and NEBNext HiFi 2x PCR Master mix. PCR cycling was conducted as follows: 72°C for 5 min, 98°C for 30 sec, followed by 14 cycles of 98°C for 10 sec and 63°C for 10 sec, and a final extension step of 72°C for 1 min. PCR products were purified using Ampure XP beads. After equimolar pooling of barcoded libraries, samples were submitted to DNA Technologies and Expression Analysis Core, University of California Davis Genome Center, for bead clean-up to remove PCR primers and fragments longer than 1,000 bp. Sequencing was performed on the Illumina HiSeq 4000 platform and produced paired-end 40 bp reads.