GSM5698762: NlaIII5plusplusnew2 : Stable population sample B2; Homo sapiens; OTHER
Genomic DNA was isolated from 5 million cells using the DNeasy kit (Qiagen). Four micrograms of genomic DNA (gDNA) was digested with NlaIII or MseI. After PCR spin column purification (Qiagen), 50 ml of eluted digested DNA was ligated using T4 DNA ligase (Roche) in a volume of 2 ml. The reaction mix was purified using spin columns and used as the template in a PCR with primers annealing to internal sequences in the gene-trap vector (59-CTGCAGCATCGTTCTGTGTT-39 and 59-TCTCCAAATCTCGGTGGAAC-39). The resulting PCR products were used for library preparation. Sequencing libraries were created using the Ion Plus Fragment Library Kit (ThermoFisher Scientific) according to manufacturer's instructions, with minor modifications: briefly, 15 ng of the PCR products were diluted with ddH2O to a final volume of 39 μl. The samples were end repaired, adaptor ligated and amplified; this was followed by 2 rounds of purification using Agencourt AMPure XP beads. Library qualities and quantities were assessed by Bioanalyzer, using the DNA High Sensitivity Kit (Agilent Technologies). The quantified libraries were pooled together in 10plex, at a final concentration of 40 pM. Templating, enrichment and chip loading was performed on an Ion Proton Chef system using the Ion PI™ Hi-Q™ Chef Kits (ThermoFisher Scientific); sequencing was performed on an Ion Proton PI™ V3 chip, with the Ion PI™ Hi-Q™ Sequencing 200 Kit, on an Ion Proton instrument (ThermoFisher Scientific), according to manufacturer's instructions.