GSM5221275: NPC H2B-Dam repl1; Mus musculus; OTHER
DNA extracted using Quick-DNA Miniprep Plus kit (Zymo Research). DamID seq was performed on H2B-Dam and H2B-Dam-NoLS Doxycycline and Shield1-induced samples adapting the previously described protocol to work with Next Generation sequencing (Vogel et al., 2007). Briefly, 500ng of gDNA previously extracted with Quick-DNA Miniprep Plus kit (Zymo Research) was digested for 4h at 37°C with DpnI (New England Biolabs) to cut methylated GATC sites. After heat inactivation for 20 minutes at 80°C, DamID adaptors (Vogel et al. 2007) were blunt-end ligated overnight at 16°C and then heat inactivated at 65°C for 10 minutes. In order to cut unmethylated GATC sequences, DNA was digested for 3h at 37°C with DpnII (New England Biolabs) and heat inactivated at 65°C for 20 minutes. Adaptor-ligated fragments were amplified using the Advantage® GC 2Polymerase mix (Clontech Takara) (primer described in Table MM, Vogel et al., 2007). DamID libraries were purified using Agencourt AMPure XP beads (Beckam Coulter). Since the size of fragments produced exceeded the one fitting the sequencing machine, the libraries were fragmented using the ds Fragmentase (New England Biolabs) to enrich the concentration of the libraries below 500bp and, afterwards, the libraries were purified again using the Agencourt AMPure XP beads (Beckam Coulter). The quantity and quality of the isolated DNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA). The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) to prepare the libraries for Illumina sequencing. Briefly, Nucleolar-DamID samples (1 ng) were end-repaired and polyadenylated. Then, Illumina compatible adapters, containing the index for multiplexing, were ligated. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA) the sequencing was performed as single end 150bp reads using the Illumina NovaSeq 6000.