title: GSM8438470: ESC, RNA, Rep3; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei isolation from in vitro cultured cells. For nuclei isolation, cells were incubated in NE1 buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1 % Triton X-100, 20 % glycerol, 1X cOmplete Proteinase Inhibitor) for 3 min on ice. Nuclei were fixed with 0.1 % formaldehyde (Thermo Scientific, 28906) in PBS for 1 min at room temperature. Formaldehyde was quenched with 74 mM glycine and nuclei were pelleted by centrifugation at 450 xg for 3 min at 4 °C. RNA isolation and RNA-seq from in vitro cultured cells. Total RNA from cultured cells were extracted using the NucleoSpin RNA Kit (Macherey-Nagel, 740955.50) according to the manufacturer's protocol. RNA-seq libraries were generated with the QuantSeq 3' mRNA-Seq Library Prep Kit FWD with Unique Dual Indices for Illumina (Lexogen, 115.384) and sequenced on an Illumina NovaSeq 6000 platform using NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) (Illumina, 20028401). All RNA-seq experiments were conducted in four independent technical replicates. 10,000 nuclei were transferred into new 0.2 mL PCR tubes and incubated for 3 h at 58 °C in 50 µL Denaturation buffer (10 mM Tris pH 8.0 (HCl), 50 mM NaCl, 0.2 % (w/v) SDS, 0.5 ng/mL proteinase K). Denaturation was stopped by purification of the tagmented DNA fragments using the MinElute Reaction Cleanup Kit (QIAGEN, 28204). DNA was eluted in 23 µL EB buffer and amplified analogously to CUT&Tag (https://www.nature.com/articles/s41467-019-09982-5) for 12-20 cycles. Appropriate number of cycles can be estimated via qPCR and will depend on epitope abundancy. Amplified sequencing libraries were one-sided size-selected using first a 0.5 X SPRI beads ratio to remove large fragments followed by purification with 1.3 X SPRI beads ratio followed by elution in 26 µL EB buffer.