title: GSM2849965: cDNA reads, ACF1, repl 1; Drosophila melanogaster; OTHER
library_construction_protocol: To map the reporter integrations, gDNA was extracted from 20*106 cells using ISOLATE II Genomic DNA Kit (Bioline) and digested with NlaIII (40 U, NEB) in buffer 4 supplemented with BSA in a total volume of 100 µl for 2 h at 37°C. The reaction was terminated by heat inactivation for 20 min at 65°C. Fragments were circularized over night at 12°C using T4 Ligase (100 U, NEB) in T4 Ligase buffer in a total volume of 1600 µl. Products were precipitated with ethanol and purified with ISOLATE II PCR and Gel kit (Bioline). Unligated fragments were removed by digestion with Plasmid-Safe DNase (20 U, Epicentre) with added ATP (final concentration 1 mM) in the manufacturer-supplied reaction buffer in a total volume of 100 µl for 5 h at 37°C. The reaction was terminated by heat-inactivation for 30 min at 70°C and purified using ISOLATE II PCR and Gel kit (Bioline). To eliminate any remaining unintegrated plasmid , products were digested with I-CeuI (5 U, NEB) in buffer 4 supplemented with BSA in s total volume of 60 µl for 2 h at 37°C. The reaction was terminated by heat-inactivation for 20 min at 65°C and purified using ISOLATE II PCR and Gel kit (Bioline). Products containing barcodes and flaking gDNA were amplified in two rounds by inverse PCR to add Illumina adapters for Illumina paired-end sequencing. For cDNA reads, RNA from 10*106 transfected cells isolated by FACS sorting was extracted with Chloroform from TRIsure-resuspended samples (Bioline). The polyadenylated fraction was isolated using the Oligotex kit (Qiagen). RNA samples were treated with RNase-free DNase I (2 U, Roche) in DNase buffer in a total volume of 20 µl for 30 min at 37°C. The reaction was terminated by adding 1 µl of 25 mM EDTA and incubation at 70°C  for 15 min. cDNA was generated using Tetro cDNA Synthesis Kit (Bioline) according to the manufacturer's instructions with a GFP-specific primer instead of OligoDT. 1/4 of total cDNA was used in a PCR reaction to add adapters for Illumina single-end sequencing. The appropriate PCR cycle number was determined by qPCR to avoid overamplification. For gDNA reads, gDNA was isolated from 10*106 unsorted TRIP cells using ISOLATE II Genomic DNA Kit (Bioline). 500 ng were used in a PCR reaction to add adapters for Illumina single-end sequencing and amplified over 25 cycles.
attributes.tag: GEO Accession
attributes.value: GSM2849965