GSM3660447: 100 nM_2.7kb_pure_clone_-dox_rp2; Mus musculus; OTHER
3x106 cells were harvested using trypsin after 18 hours of induction with tamoxifen +/- doxycyclin. Genomic DNA was extracted using the Qiagen blood and tissue kit adding RNaseA in step 1. Genomic DNA was eluted in 80ul ddH2O. DNA concentration was measured using the Qbit DNA Broad Range kit Genomic DNA (100ng input) was treated with Shrimp Alkaline Phosphatase treatment (NEB, 1U), followed by DpnI digestion (ThermoFisher Scientific, 10U), A-tailing (0.6mM final dATP, 5U Klenow exo-, ThermoFisher Scientific), and UMI adapters ligation (30U T4 DNA ligase, PEG4000, ThermoFisher Scientific) performed within the same tube and buffer (Tango 1X, ThermoFisher Scientific) by heat inactivating each enzymatic step followed by adjustment with the reagents required for the next step, Ligation reactions were treated with Exonuclease I (20U, ThermoFisher Scientific ) then purified using AMPureXP beads (1:0.8 ratio, Agencourt) and the second sequencing adapter was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (10 cycles) using 1U of Phusion polymerase, 2μl 10 μM DAM_UMIindex_PCR was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (10 cycles) using 1U of Phusion polymerase, 2μl 10 μM DAM_UMIindex_PCR.
GEO Accession
GSM3660447