Nimblegen seqcap ez accessory kit v2

5′ biotinylated probes (see Table S2) were designed using the online tool by the Hughes lab (CapSequm, http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi) to be 70-120bp long and two probes for each promoter of interest. The probes were pooled at 2.9nM each. Samples were captured twice and hybridizations were carried out for 72h and for 24h for the first and the second captures, respectively. To even out capture differences between tubes, libraries were pooled prior to hybridization. For Control, SCC1^DEG, RING1B^DEG and SCC1^DEG RING1B^DEG, 1.5μg of each replicate was individually hybridized and then pooled for the second round of hybridization. CTCF ± AUX were multiplexed prior to the first capture at 2 μg each. Hybridization was carried out using Nimblegen SeqCap (Roche, Nimblegen SeqCap EZ HE-oligo kit A, Nimblegen SeqCap EZ HE-oligo kit B, Nimblegen SeqCap EZ Accessory kit v2, Nimblegen SeqCap EZ Hybridization and wash kit) following manufacturer’s instructions for 72 h followed by a 24 h hybridization (double Capture). The captured library molarity was quantified by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche) and sequenced on Illumina NextSeq 500 platform for three biological replicates.

The probes described in Supplementary Table S1 were pooled at 2.9 nM each. Samples were captured twice and hybridizations were carried out for 72 h and for 24 h for the first and the second captures, respectively. To even out capture differences between tubes, libraries were pooled prior to hybridization at 1.5 μg each. Hybridization was carried out using Nimblegen SeqCap (Roche, Nimblegen SeqCap EZ HE-oligo kit A, Nimblegen SeqCap EZ HE-oligo kit B, Nimblegen SeqCap EZ Accessory kit v2, Nimblegen SeqCap EZ Hybridization and wash kit) following manufacturer's instructions. The captured library molarity was quantified by quantitative polymerase chain reaction (qPCR) using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche) and sequenced on Illumina NextSeq 500 platform for 4 (ESC Fbxl19-CxxC^f/f), 3 (Med13/13l^f/f) or 2 (RA Fbxl19-CxxC^f/f) biological replicates.

5′ biotinylated probes were designed using the online tool by the Hughes lab (CapSequm) to be 70-120bp long and two probes for each promoter of interest. The probes were pooled at 2.9 nM each. Samples were captured twice and hybridizations were carried out for 72 h and for 24 h for the first and the second captures, respectively. To even out capture differences between tubes, libraries were pooled prior to hybridization at 1.5 μg each. Hybridization was carried out using Nimblegen SeqCap (Roche, Nimblegen SeqCap EZ HE-oligo kit A, Nimblegen SeqCap EZ HE-oligo kit B, Nimblegen SeqCap EZ Accessory kit v2, Nimblegen SeqCap EZ Hybridization and wash kit) following manufacturer’s instructions for 72 h followed by a 24 h hybridization (double Capture). The captured library molarity was quantified by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche) and sequenced on Illumina NextSeq 500 platform as 80 bp paired-end reads for three biological replicates.