title: GSM8149139: 006_HEK293T_input_rep2; Homo sapiens; OTHER
library_construction_protocol: KARR-seq: Live cells or homogenized tissue samples were treated with N3-kethoxal, cells were then lysed and RNA inproximity is crosslinked by dendrimers. The sample was then treated with proteinase K and Dnase. RNA was extracted using phenol chloroform.  smallRNA-seq: Total RNA was extracted using TRIzol, and small RNA fraction was isolated using the mirVana™ miRNA Isolation Kit (Invitrogen, AM1561). KARR-seq: 10-50 ng of ligated RNA was used for reverse transcription (RT) using the RT reagents in the SMARTer Stranded Total RNA-seq kit v2 – pico input mammalian (Takara, 634413). After RT, 2 µL of RNase H (NEB M0297S) was added to each sample, which was then incubated for 20 minutes at 37°C. The reaction was quenched by heating inactivation for 5 minutes at 70°C. Subsequently, cDNA was purified using the DNA Clean & Concentrator-5 kit. 10% of the cDNA was saved as an input sample, while the remaining cDNA was used for snoRNA enrichment. After streptavidin beads pulldown, the resulting cDNA was subjected to library construction using the SMARTer Stranded Total RNA-seq kit v2 – pico input mammalian (Takara, 634413).   Small RNA-seq: Small RNA fractions were isolated using the mirVana™ miRNA Isolation Kit (Invitrogen, AM1561) according to the manufacturer's protocol. The resulting small RNA fraction was subsequently used for sequencing library construction using the NEBNEB Next Small RNA Library Prep Set for Illumina (NEB E7330S) according to the manufacturer's protocol.