GSM5733569: Whole Genome Sequencing of HoxA -/- A17iCre mESCs; Mus musculus; Rattus norvegicus; OTHER
Plasmids were purified from small scale bacterial cultures (5-10 mL) using alkaline lysis. gDNA was extracted from mESC cell pellets using Qiagen DNA mini kit. Illumina sequencing libraries for BACs were generated from 100-200 ng BAC DNA using NEBNext Ultra II FS DNA Library Prep Kit (New England Biolabs E7805S) according to the manufacturer's protocol. Whole genome sequencing and capture sequencing libraries were prepared as follows: 1 µg of DNA was sheared to ∼500 to 900 bp in a 96-well microplate using the Covaris LE220 (450 W, 10% Duty Factor, 200 cycles per burst, and 90-s treatment time). Sheared DNA was purified using the DNA Clean and Concentrate-5 Kit (Zymo Research D4013), and the concentration was measured on a NanoDrop instrument (Invitrogen). DNA fragments were end-repaired with T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase (New England Biolabs M0203S, M0210S and M0201S, respectively), and A-tailed using Klenow (3′-5′ exo-; New England Biolabs M0212L). Illumina-compatible adapters were subsequently ligated to DNA ends, and DNA libraries were amplified with KAPA 2X Hi-Fi Hotstart Readymix (Roche). Biotinylated baits for capture sequencing were prepared from assemblon BACs using nick translation