GSM8940195: pFC9, Add SSB, Untranscribed; synthetic construct; Bisulfite-Seq
Negatively supercoiled plasmid substrates were purified by alkaline lysis protocol followed by column cleanup (Qiagen) and eluted into either TE or EB buffer. Single-Molecule R-loop Footprinting (SMRF-seq) samples were amplified with a combination of forward and reverse primers that each incorporated a unique barcode. The combination of the forward and reverse primer barcodes uniquely identifies each sample within its specific flowcell. Primer binding sequences were designed in Primer3 (10) software and barcodes were generated using a custom Python script. Samples were amplified using Q5U® Hot Start High-Fidelity DNA Polymerase (NEB M0515S) for 25-30 cycles according to the manufacturer's online protocol. 1-3 μl of bisulfite converted substrate was used for each reaction. In the case that the yield of one reaction was not sufficient, additional reactions using the original bisulfite converted sample were performed and then pooled. The target amplicon size was then purified via agarose gel extraction using BioBasic gel extraction kit (BS654). Sample concentration was quantified via agarose gel electrophoresis against 1kb linear DNA ladder (Thermo Fisher SM0311). SMRT cell PacBio libraries were constructed by the UC Davis DNA Technologies core according to manufacturer's instructions and sequenced on a PacBio Sequel II instrument. Additional sequencing was performed at the UC Berkeley QB3 sequencing core on a PacBio Revio instrument.