title: GSM9084193: L3455-GM18278_ssSMF; Homo sapiens; OTHER
library_name: GSM9084193
library_construction_protocol: Cells were centrifuged at 1,000 g for 2 minutes, then resuspended in 1 mL 1xPBS with 27 uL 37% formaldehyde added (to a final concentration of 1%). Crosslinking was carried out for 15 minutes at room temperature on a rotator, then quenched with 100 uL 2.5 M glycine solution. Crosslinked cells were centrifuged at 1,000 g for 2 minutes, resuspended in 1 mL 1xPBS, and centrifuged again. Permeabilization was carried out either by resuspending cells in Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) and incubating on ice for 10 minutes, then centrifuging for 2 minutes at 1,000 g. Cells (200,000 cells were used as input) were then resuspended in 1 mL of 1xAPOBEC Buffer  (from NEB EM-seq kit: NEB #E7120). Deamination treatment was carried out by adding 2 uL RNAse A and 10 uL PURExpress CseDa0, then incubating at 37C for 3 hours with shaking at 800 rpm in a Thermomixer. After deamination, cells were again pelleted by centrifugation at 1,500 g for 2 minutes, then resuspended in 200 uL IP Elution Buffer (1% SDS, 0.1 M NaHCO3); 2 uL Proteinase K were added, and reverse crosslinking was carried out overnight at 65C. DNA was then isolated using the MinElute kit (Qiagen) using 600 uL PB buffer and following the manufacturer's instructions, eluting in 52 uL EB buffer. For single-stranded libraries construction, SMF DNA was first sheared using a Covaris E220 instrument to an average size of 400-500 bp, then the xGen Methyl-Seq DNA Library Prep Kit (IDT)) was used to generated Illumina-compatible libraries, which in turn were converted into Ultima-compatible libraries using a constant primer (CTGTGTGCCTTGGCAGTCTCAGCTCAGACGTGTGCTCTTCCGATC*T) and an index primer (CCATCTCATCCCTGCGTGTCTCCGACTGCACXXXXXXXXXXXGATCTACACGACGCTCTTCCGATC*T, where the portion indicated as ``XXXXXXXXXXX'' corresponds to the index sequences). For each library, ~10 ng were used as input, diluted in a 20 uL volume, then mixed with 2.5 uL of each primer and 25 uL of 2x NEB Next High Fidelity PCR Master Mix (NEB Cat \# M0541L). Libraries were amplified using initial denaturation at 98C for 45 seconds, then 7 cycles of 15 seconds at 98C, 30 seconds at 60C, and 60 seconds and 72C, followed by final extension for 10 minutes at 72C.
identifiers.namespace: GEO
identifiers.id: GSM9084193_r1