GSM7912214: E8.5 embryo, WT, ONT adaptive sampling; Mus musculus; OTHER
Genomic DNA (gDNA) was isolated using the salt-extraction method. Briefly, tissues were lysed in cell lysis buffer (50 mM Tris-HCl (pH 8), 4 mM EDTA (pH 8), 2% SDS) plus Proteinase K (390973P, VWR), and incubated at 55°C overnight. Cell lysate was treated with RNaseA (EN0531, Thermo) at 37°C for 1 h. Saturated NaCl buffer was added, followed by addition of isopropanol to precipitate gDNA and washing with 70% EtOH. gDNA was dissolved in water. DNA concentration was determined by Qubit using the Qubit High Sensitivity Kit and size was determined on a Femto Pulse System (Agilent Technologies). ONT sequencing libraries were prepared using the Genomic DNA ligation kit (SQK-LSK114) and sequenced using the vendors instructions. Briefly, 600ng gDNA was used as input and yielded approximately 20fmol of library to load on a PromethION Flowcell (FLO-PRO114M) and sequenced on a PromethION 24 device (A100). Adaptive Sampling in MinKNOW (v22.10.7) was enabled during the sequencing of these samples using fast basecalling and a BED file was provided for the regions of interest while mm10 of the mouse genome was used as a reference for the enrichment. After 20h the run yielded around 400k reads which accounts for 3.53Gb with a N50 of 28kb.