GSM2344445: WT_AAM_5h_R1; Mus musculus; OTHER
The original ATACseq protocol {Buenrostro, 2013 #118} was modified according to Lara-Astiaso et al. {Lara-Astiaso, 2014 #122}. Briefly, 50 X 103 cells were pelleted by centrifugation and re-suspended in 25 µl of cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Igepal CA-630). Nuclei were pelleted by centrifugation for 20 min at 500g, 4 ºC.  The supernatant was discarded and nuclei were re-suspended in 25 µl of reaction buffer containing 1 µl of Tn5 transposase (made in house) and 5x transposase buffer (50mM Tris-HCl, pH 8.4 and 25mM MgCl2). The reaction was incubated at 37ºC for one hour. Then 5 µl of clean up buffer (900mM NaCl, 300mM EDTA), 2ul of 5% SDS and 2 µl of Proteinase K (20 µg/µl) (New England Biolabs) were added and incubated for 30 min at 40 ºC. Tagmented DNA was isolated using SPRI beads (2x). For library amplification, two sequential PCR reactions were performed in order to enrich small tagmented DNA fragments. We used 2 µl of indexing primers and KAPA HiFi HotStart ready mix. After the first PCR, fragments smaller than 600 bp were isolated using SPRI cleanup and a second PCR was performed using the same conditions in order to obtain the final library. DNA concentration was measured with a Qubit fluorometer (Life Technologies) and library quality was analyzed using a Bioanalyzer (Agilent Technologies). Libraries where sequenced on an Illumina HiSeq 2000.