GSM2175815: RICCexpt3-300gy-lib7; Homo sapiens; OTHER
Cells were lysed in agarose plugs by incubating with 0.4M EDTA pH 8.0, 2% (w/v) Sarkosyl, 0.5 mg/mL Proteinase K at 50˚C for 24-48 hours, then washed 3x with TE(10 mM Tris, 1 mM EDTA, pH 8), for 1 hour each wash at room temperature), 2x with TE containing 0.1 mg/mL RNase A at 37˚C, and 2x with TE at room temperature. Agarose-embedded genomic DNA was treated with shrimp alkaline phosphatase, then the enzyme was inactivated with lysis buffer (0.4M EDTA pH 8, 2% (w/v) Sarkosyl, 0.5 mg/mL Proteinase K) at 50˚C for 18 hours. Samples were washed 5x with TE at room temperature, then treated with ~0.4 U/µL T4 polynucleotide kinase in 70 mM Tris, 10 mM MgCl2, 5 mM DTT, 1 mM spermidine and 5 µM ATP for 30 min at 37˚C, and washed 4x with TE.  ssDNA fragments were eluted from agarose plugs by passive diffusion into TE following thermal denaturation at 95˚C and an ice quench. A truncated version of Illumina TruSeq adapters with random hexamer overhangs were ligated to concentrated ssDNA fragments using T4 DNA ligase (50 pmol each adapter per ssDNA from 1.5 million cells). After removing excess adapters, the resulting library was made double stranded using Taq-B polymerase (Enzymatics), size selected on an agarose gel, amplified and barcoded via PCR, and size selected again on an agarose gel.
GEO Accession
GSM2175815