Instrument: Illumina HiSeq 2500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: High molecular weight genomic DNA was extracted fromCD3+ T-cell from human cord blood. Custom adapters were created for HELP-tagging, referred to as AE and AS Index. As well as an Illumina adapter sequence, adapter AE contains an EcoP15I recognition site and a T7 promoter sequence, and adapter AS contains index sequences for multiplexing . Adapter AS contains an Illumina sequencing primer sequence. Five micrograms of genomic DNA were digested with HpaII in separate 200 μl reactions and purified by phenol/chloroform extraction followed by ethanol precipitation. The digested genomic DNA was ligated to adapter AE using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 3 μg of HpaII-digested DNA, 0.1 μl of Adapter AE (1 μM), 3 μl of Quick Ligase in a final volume of 50 μl). After AE ligation, the products were purified using AMpure XP (Agencort), then digested with EcoP15I (NEB). The restriction fragments were end-repaired to inhibit to dimerization of adapters, and tailed with a single dA, at the 3’ end. After the dA tailing reaction, adapter AS was ligated to the dA-tailed fragments using an NEB Quick Ligation Kit (25 μl of 2x Quick ligase buffer, 2.5 μl of Adapter AS (10 μM), 2.5 μl of Quick Ligase in a final volume 50 μl). After ligation, products were purified using the MinElute PCR purification kit (Qiagen) and in vitro-transcribed using MEGAshortscript (Ambion). Following in vitro transcription, products were purified with an RNeasy clean-up kit (Qiagen) before reverse transcription was performed using the SuperScript III kit (Invitrogen). The first strand cDNA produced was used as a template for PCR using the following conditions: 96°C for 2 minutes, then 18 cycles of 96°C for 15 seconds and 72°C for 15 seconds followed by 5 minutes at 72°C for the final extension. After PCR, the library was purified using a QIAQuick PCR clean-up kit (Qiagen).