GSM7025875: WT_BLISS_rep1; Mus musculus; OTHER
We followed the suspension-cell BLISS protocol. Cells were fixed in 4% paraformaldehyde (15710, Electron Microscopy Sciences) for 10 min at room temperature on a roller shaker. Formaldehyde was quenched with glycine. Fixed cells were then washed twice using ice-cold PBS. For permeabilization, fixed cells were subjected to two sequential lysis using lysis buffer 1 (10mM Tris-HCl, 10mM NaCl, 1mM EDTA, 0.2% Triton X-100, pH 8) and lysis buffer 2 (10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 0.3% SDS, pH 8). After permeabilization, DSB ends were blunted using the Quick Blunting Kit (E1201L, NEB) for 1 h at room temperature, which was followed by in situ DSB ligation of BLISS adapters using T4 DNA ligase (M0202M, NEB) for ~16 h (overnight) at 16°C. On Day 2, genomic DNAs were reverse crosslinked and extracted using DNA extraction buffer (10mM Tris-HCl, 100mM NaCl, 50mM EDTA, 1% SDS, 1mg/mL Proteinase K, pH 8) at 55°C for ~18 h (overnight) with shaking. On Day 3, DNA was purified and then sonicated in 1 x TE buffer using the Covaris M220 ultrasonicator with the following setting: peak incident power 50, duty factor 10%, cycles per burst 200, time 70s. DNA was then purified by Agencourt AMPure XP beads. DNA was in vitro transcribed using the MEGAscript T7 Transcription Kit (AM1333, ThermoFisher) at 37°C for ~14 h (overnight). On Day 4, genomic DNA was removed using DNase I (RNase-free) (AM2222, ThermoFisher) followed by RNA purification using Agencourt RNAClean XP beads (Beckman Coulter). Libraries were built via the following steps: Illumina RA3 adapter ligation using T4 RNA Ligase 2, truncated KQ (M0373S, NEB) at 25°C for 2 h; reverse transcription using SuperScript III Reverse Transcriptase (18080044, ThermoFisher) at 50°C for 1 h; library indexing and amplification using NEBNext High-Fidelity 2x PCR Master Mix (M0541S, NEB); DNA purification and size-selection (300-800bp) using Agencourt AMPure XP beads. All primers and adapters were custom synthesized by Integrated DNA Technologies (IDT) based on sequences in TruSeq Small RNA Library Preparation kit (Illumina).