alias: GSM3532091
title: GSM3532091: SetD_FX_nt; Homo sapiens; OTHER
library_construction_protocol: 10E6 PBS-washed cells were embedded in 0.5% Incert low-melting point agarose in PBS and cast into plugs (0.5cmx1cmx1.5mm).  Plugs were then incubated in  6 ml lysis buffer (0.5M EDTA, 1% Sarkosyl, 200ug/ml Proteinase K) at 50 degree celcius for 48 h, with a buffer exchange at 24 h.  Plugs were then washed three times, for 1 h each, with 6 ml TE at room temperature. The DNA in TE-washed agarose plugs was then in-gel end-labeled with biotinylated dNTP mix (1 mM dTTP, dCTP, dGTP, 0.84 mM dATP, 0.16 mM Biotin-14-dATP) using End-It Kit (Epicentre). Plugs were then treated with beta-Agarase (NEB) to digest agarose and release DNA. DNA sample was then sonicated using a Covaris M220 using the snap-cap DNA 300bp shearing protocol.  DNA was then processed using a PCR Cleanup Kit (Qiagen) and run on agarose gel to verify the fragmentation pattern of DNA and quantified on a Nanodrop. 10-11 μg of DNA was then end-repaired using a regular End-It kit and purified by the PCR Clean-up Kit.  The DNA was then A-tailed by A-tail Kit or exo-Klenow (NEB) and purified by PCR Clean-up kit, followed by quantification on a Nanodrop.  M270 Dynabeads (Life Technologies) were used to purify biotinylated DNA.  DNA bound to beads was calculated by measuring the quantity of DNA in the flow through.  DNA-bound beads were then resuspended in a T4-ligase mix containing Illumina adaptors.  400 ng of DNA bound to beads was used for PCR amplification using KAPA Hotstart Ready Mix (KAPA).  Each sample was given a specific index primer for multiplexing.  PCR product was then run on agarose gel to verify amplification and quantity.  AMPure beads (Agencourt) were used to remove free adaptors and the final product was analyzed on agarose gel. Break-seq libraries were sequenced on Illumina Hi-Seq 2500 with 100 or 150 bp paired-end reads, followed by Break-seq data analysis. 10E6 PBS-washed cells were embedded in 0.5% Incert low-melting point agarose in PBS and cast into plugs (0.5cmx1cmx1.5mm).  Plugs were then incubated in  6 ml lysis buffer (0.5M EDTA, 1% Sarkosyl, 200ug/ml Proteinase K) at 50 degree celcius for 48 h, with a buffer exchange at 24 h.  Plugs were then washed three times, for 1 h each, with 6 ml TE at room temperature. The DNA in TE-washed agarose plugs was then in-gel end-labeled with biotinylated dNTP mix (1 mM dTTP, dCTP, dGTP, 0.84 mM dATP, 0.16 mM Biotin-14-dATP) using End-It Kit (Epicentre). Plugs were then treated with beta-Agarase (NEB) to digest agarose and release DNA. DNA sample was then sonicated using a Covaris M220 using the snap-cap DNA 300bp shearing protocol.  DNA was then processed using a PCR Cleanup Kit (Qiagen) and run on agarose gel to verify the fragmentation pattern of DNA and quantified on a Nanodrop. 10-11 microgram of DNA was then end-repaired using a regular End-It kit and purified by the PCR Clean-up Kit.  The DNA was then A-tailed by A-tail Kit or exo-Klenow (NEB) and purified by PCR Clean-up kit, followed by quantification on a Nanodrop.  M270 Dynabeads (Life Technologies) were used to purify biotinylated DNA.  DNA bound to beads was calculated by measuring the quantity of DNA in the flow through.  DNA-bound beads were then resuspended in a T4-ligase mix containing Illumina adaptors.  400 ng of DNA bound to beads was used for PCR amplification using KAPA Hotstart Ready Mix (KAPA).  Each sample was given a specific index primer for multiplexing.  PCR product was then run on agarose gel to verify amplification and quantity.  AMPure beads (Agencourt) were used to remove free adaptors and the final product was analyzed on agarose gel. Break-seq libraries were sequenced on Illumina HiSeq 2500 with 100 or 150 bp paired-end reads, followed by Break-seq data analysis.
identifiers.namespace: GEO
identifiers.id: GSM3532091