GSM7520263: mESC_WT_m6A-CT1; Mus musculus; OTHER
For bulk m6A-CT library preparation, E14, EPSC, MEF and HEK293T nuclei were isolated with the following conditions: 1 min on ice in 0.020 % Triton X-100 for E14, 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF, 0.02 % NP-40 for HEK293T. Aliquots of nuclei (5,000–100,000 nuclei) were bound to pre-activated Concanavalin A (Con-A) Conjugated Paramagnetic Beads (EpiCypher, #21-1411) and used in assay. All wash steps were performed on a magnetic separation rack. Undirected cDNA/RNA hybrid tagmentation for INPUT RNA was carried out as described previously. Briefly, nuclei were resuspended in 50 μL Reverse Transcription & Tagmentation Buffer, and incubated for 1 hour at 37 °C. 2.5 μL pAG-Tn5 was then added and vortexed briefly, and incubated for 1 hour at 37 °C.  Antibody directed tagmentation for m6A-CT was carried out as  same as sc-m6A-CT using 1 μg anti-m6A antibody (Synaptic Systems, #202003). When tagmentation was done, 54.5 μL of 0.5 M EDTA, 1 μL of 20 mg/mL Proteinase K and 5.5 μL of 10% SDS were sequentially added to the sample and incubated at 58 °C for 1 hour. An equal amount of Phenol-chloroform-isoamyl alcohol 25:24:1 (PCI), pH 6.7 was added to each sample, and PCI extraction followed by ethanol precipitation were performed. To perform gap-filling, the purified RNA/DNA samples were mixed with 0.32 U/μL Bst 3.0 DNA Polymerase (New England Biolabs) and Q5 High-Fidelilty 2× PCR Master mix (New England Biolabs), and incubated for 5 min at 58 °C, 5 min at 72 °C, and then at 5 min at 80 °C to inactivate Bst 3.0. A qPCR cycle check can be performed at this stage using 1:100 templates. A successful experiment can be identified by comparing cycle threshold values to IgG-CT. A universal i5 and a uniquely barcoded i7 primer, and Q5 HiFi 2× PCR Master mix were used to amplify libraries with the following cycling conditions: 45 sec at 98 °C, followed by 14-21 cycles of 15 sec at 98 °C and 10 sec at 60 °C, with a final extension at 1 min at 72 °C. Post-PCR clean-up was performed by 0.5× right-side followed by 1.2× left-side SPRI beads (Beckman Counter) selection.