Biotin 14 datp

In nucleus, Hi-C library generation was performed as previously described [21 (link)]. Briefly, fixed adult liver tissue from three biological replicates at ZT0 0, 6, 12, and 18 was sieved through a 70-μM cell strainer and dounce homogenized in 10 ml of ice-cold lysis buffer with a tight pestle for a total of 30 strokes on ice. Nuclei were washed and permeabilized with 0.3% SDS for 45 min at 37 °C and then incubated overnight with HindIII at 37 °C, DNA ends were labeled with biotin-14-dATP (Life Technologies) in a Klenow end-filling reaction and ligated in nuclei overnight. DNA was purified by phenol-chloroform, and the concentration was measured using Quant-iT PicoGreen (Life Technologies). A total of 10 μg of DNA was sheared to an average size of 400 bp using a Covaris machine and following the manufacturer’s instructions. The sheared DNA was end-repaired, adenine tailed, and subject to a double size selection using AMPure XP beads to isolate DNA ranging from 250 to 550 bp in size. Ligated fragments marked by biotin-14-dATP were pulled-down using MyOne Streptavidin C1 DynaBeads (Invitrogen) and ligated to paired-end adaptors (Illumina). The in nucleus Hi-C libraries were amplified using the PE PCR 1.0 and PE PCR 2.0 primers (Illumina) using 6–9 PCR amplification cycles as required.

The non-cloned 5S rDNA and U snDNAs sequences and telomeric probes were labeled through PCR using digoxigenin-11-dUTP (Roche, Mannheim, Germany). Plasmids containing the 18S rRNA gene or H3 histone gene and the C₀t-DNA fraction were labeled via nick translation using biotin-14-dATP (Invitrogen, San Diego, CA, USA). Single or two-color FISH of mitotic cells was performed according to Pinkel et al. [51 (link)], with modifications [45 (link)]. Fiber-FISH was conducted as described in de Barros et al. [52 ] and Camacho et al. [53 ] using suspensions of testis cells. Probes labeled with digoxigenin-11-dUTP were detected using rhodamine-conjugated anti-digoxigenin (Roche), and probes labeled with biotin-14-dATP were detected using Streptavidin Alexa Fluor 488-conjugated (Invitrogen).
The preparations were counterstained using 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) and were mounted in Vectashield (Vector, Burlingame, CA, USA). The chromosomes and signals were observed using an Olympus microscope BX61 equipped with a fluorescence lamp and the appropriate filters. Grey-scale images were captured using a DP70 cooled digital camera and were processed using Adobe Photoshop CS2 software.

The probes for 18S rDNA and H3 histone genes were labeled by nick-translation using biotin-14-dATP (Invitrogen, San Diego, CA, USA), while the 5S rDNA, U2 snDNA and telomeric probes sequences were labeled through PCR with digoxigenin-11-dUTP (Roche, Mannheim, Germany). Single and two color FISH experiments were performed as in Cabral-de-Mello et al. [20 (link)]. Probes labeled with digoxigenin-11-dUTP were detected using anti-digoxigenin-rhodamine (Roche), and probes labeled with biotin-14-dATP were identified using streptavidin, alexafluor 488 conjugate (Invitrogen). The preparations were counterstained using DAPI and mounted using Vectashield (Vector, Burlingame, CA, USA). The FISH results were documented using an Olympus microscope BX61 equipped with a fluorescence lamp and appropriate filters coupled to DP70 cooled digital camera. The images were merged and optimized for brightness and contrast using Adobe Photoshop CS2 software.