SMF-ONT S2 clamp RNAi knockdown
Collection of 2 biological replicates of SMF-ONT performed in S2 cell incubated for 72hrs with RNAi targeting Clamp or LacZ (ctrl) transcripts.  The footprinting protocol was adapted from Kleinendorst and Barzaghi et al., 2021 and optimised for long-read sequencing and high weight DNA extraction. In summary, 2.5 million intact nuclei per SMF reaction have been treated with successive incubation with GpC (M.CviPI) and CpG (M.SssI) methyltransferase enzymes. Footprinted DNA was extracted using the Quick-DNA HMW MagBead kit following the manufacturer protocol and using wide-bore tips. Nanopore ligation library (LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using promethION flow cells for the R10 chemistry (LSK114).  The data have been analysed using the SMF-ONT nextflow pipeline (https://git.embl.de/grp-krebs/nf-smfont) using custom methylation call models.
Collection of 2 biological replicates of SMF-ONT performed in S2 cell incubated for 72hrs with RNAi targeting Clamp or LacZ (ctrl) transcripts.  The footprinting protocol was adapted from Kleinendorst and Barzaghi et al., 2021 and optimised for long-read sequencing and high weight DNA extraction. In summary, 2.5 million intact nuclei per SMF reaction have been treated with successive incubation with GpC (M.CviPI) and CpG (M.SssI) methyltransferase enzymes. Footprinted DNA was extracted using the Quick-DNA HMW MagBead kit following the manufacturer protocol and using wide-bore tips. Nanopore ligation library (LSK114) preparation has been performed using 1µg of purified footprinted HWM DNA. Sequencing has been performed using promethION flow cells for the R10 chemistry (LSK114).  The data have been analysed using the SMF-ONT nextflow pipeline (https://git.embl.de/grp-krebs/nf-smfont) using custom methylation call models.