GSM4568567: B_HeLa_S1_L001_R1_HWGLVBCXY_V1; Homo sapiens; OTHER
Ten million HeLa cells were resuspended in PBS buffer and mixed with 1% low melting point agarose in PBS buffer at 40°C. Cell suspension was mixed with liquid paraffin at 40 °C and vigorously shaken by hand for one minute, until emulsion was formed. The emulsion was then poured into ice-cold PBS buffer and the mixture was stirred for several minutes. Agarose bead suspension was gently centrifuged (200g, 10 min), paraffin layer was removed and agarose bead pellet was washed 3 times with TE buffer. Beads were washed with ES buffer (1% Sarkosyl, 25 mM EDTA, pH 8.0), resuspended in ES with 50 µg/mL Proteinase K and incubated overnight at 50 °C. After incubation, the beads were washed with TE + 0.1 mM PMSF and twice with TE. Next, the beads were washed in 1× Blunting Buffer (NEB), followed by DNA ends blunting using Quick Blunting kit (NEB) for 2 h and then washed twice with TE. The beads were subsequently washed with dA-Tailing Reaction Buffer (NEB) and DNA ends were A-tailed using NEBNext® dA-Tailing Module for 80 min. Next, the beads were washed with T4 ligation buffer and then resuspended in T4 ligation buffer with 100 nM P5 adapter and T4 ligase (NEB) and incubated overnight at 16 °C. After ligation, the beads were washed once with TE, and encapsulated DNA was initially sonicated using Covaris S220. Total DNA was isolated using Zymoclean™ Large Fragment DNA Recovery Kit (Zymo Research) and once again fragmented by sonication to create ~400 bp fragments. Labeled fragments were captured by streptavidin beads (Invitrogen), blunted and A-tailed using NEBNext® Ultra™ End Repair/dA-Tailing, then ligated to a P7 adapter. The resulting circular DNA was then linearized by USER (NEB) digestion and amplified and indexed by PCR using Illumina PCR primers. Quality and quantity of the resulting libraries were assessed on 2100 Bioanalyzer using HS DNA Kit (Agilent), and on Qubit 2.0 Fluorometer using Qubit dsDNA HS Assay Kit (Life Technologies). The libraries were sequenced (1x61 bp) on Illumina HiSeq2500 platform, according to the modified experimental and software protocols for generation of high-quality data for low-diversity samples, as previously described (PMID: 30393778).
GEO Accession
GSM4568567