title: GSM3671440: SuRE44, library1, transfection in HEPG2, rep1; 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. PolyA RNA was divided into 10 µl reactions containing 500 ng RNA and DNase treated with 10 units for 30 minutes (#04716728001; Roche) and DNase was inactivated by addition of 1µl of 25mM EDTA and incubation at 70ºC for 10 minutes. Next, cDNA was produced by first adding 1 µl of 10 mM gene specific primer targeting the GFP ORF (247JvA) and 1 µl dNTP (10mM each) and incubating for 5 minutes at 65°C. Then 4 µl of RT buffer, 20 units RNase inhibitor (#EO0381; ThermoFisher Scientific), 200 units of Maxima reverse transcriptase (#EP0743; ThermoFisher Scientific) and 2.5 µl water was added and the reaction was incubated for 30 minutes at 50°C followed by heat-inactivation at 85° for 5 minutes. Per biological replicate, 20-30 reactions were done in parallel. Each 20 µl reaction was then PCR amplified (1' 96 °C, 20x(15'' 96 °C, 15'' 60 °C, 15'' 72 °C)) in a 100 µl reaction with MyTaq™ Red Mix and primers 151AR (containing the S1 and p5 adapter) and (index variants of) 211JvA (containing the S2, index and p7 adapter) for 21 cycles. Reactions were then pooled and 500 µl was purified using CleanPCR beads (#CPCR-0050; GC biotech) and subjected to single read 50 bp high throughput sequencing on an Illumina HiSeq2000 or HiSeq2500.
attributes.tag: GEO Accession
attributes.value: GSM3671440