title: GSM2262568: iPCR, sequencing lane 1; Homo sapiens; OTHER
library_construction_protocol: To find out which barcodes were associated with which genomic fragments, we digested 4 µg SuRE library with I-CeuI (#R0699S; NEB), followed by magnetic bead purification (1:1 ratio beads:DNA solution). Of this, 2 µg was self-ligated overnight at 16 °C in a total volume of 2 ml (#10799009001 Roche), and purified using phenol-chloroform extraction and isopropanol precipitation. To reduce the size of the genomic fragments this material was digested for 1 hour with 10 units of frequent cutter Nla III (#R0125S; NEB) or 10 units of HpyCH4V (#R0620L; NEB), bead purified and self-ligated again in a final volume of 1 ml. This material was purified by phenol-chloroform extraction and isopropanol precipitation, treated with 25 units of Plasmid-Safe™ ATP-Dependent DNase for 1 hour and purified again with phenol-chloroform and isopropanol precipitation. To facilitate PCR, the resulting mini-circles were linearized by digesting with I-SceI (#R0694S; NEB) in a volume of 25 µl. Finally, 10 cycles of PCR (1¢ at 98°C, 10x(15¢ at 98°,15¢ at 60°, 20¢ at 72°)) with Phusion high-fidelity DNA Polymerase (#M0530L; NEB) were performed on 2.5 µl of the I-SceI digested material using primers 151AR (containing the S1 and p5 adapter) and (index variants of) 117JvA (containing the S2, index and p7 adapter). The PCR product was bead purified and subjected to high-throughput paired-end sequencing on an Illumina MiSeq, HiSeq2000 or HiSeq2500.
attributes.tag: GEO Accession
attributes.value: GSM2262568