GSM2752320: Female_Alb4C_Repl3_8pool_G118_M11; Mus musculus; OTHER
Single cell suspensions were generated by passing dounce homogenized tissue through a 40 micron filter. Crosslinking was then performed with 1% formaldehyde for 10 minutes. This represents a pool of eight independent PCR reactions. Livers were harvested and sonicated according to two strategies indicated per sample. 10M Crosslinked nuclei were processed as described previously (van de Werken et al 2012, Splinter et al 2012). DpnII was used as the primary restriction enzyme and Csp6I was used as the secondary. All ligations and disgestions were overnight (minimum 16 hr), and were verified by running a small aliquot in a 1% agarose gel. Samples were amplified for 25 cycles of inverse PCR using a custom primer pair containing sequence specific to the promoter of Albumin as well as a partial adaptor sequence to facilitate barcoding in a secondary PCR reaction. To reduce the impact of PCR domination, samples represent pools of 2 or 8 independent (but otherwise identical) inverse PCR reactions as indicated. The reading primer was anchored directly at the DpnII cutsite, while the nonreading primer was positioned ~75bp from the Csp6I site (Reading primer: ACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTAAGTATGGTTAATGATC; Non-reading primer: GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCTTTGTCTCCCATTTGAG). The samples were barcoded using New England Biolabs NEBNext Multiplex Oligos for Illumina (#E7335) with 5 rounds of additional PCR per the manufacturer's instructions.
GEO Accession
GSM2752320