title: GSM3206948: NIK (Nucleolus) Target 1; Homo sapiens; OTHER
library_construction_protocol: To labeled and unlabeled (controls) HEK 293T cells in 10-cm plates, we added ~1 ml DPBS containing 5 mM Trolox and 10 mM sodium ascorbate, as well as ~4 ul Ribolock RNAse inhibitor (Thermo Fischer Scientific). The cells were then scrapped off 10-cm plates using cell lifters (Corning), transferred to 2-ml Eppendorf tubes, and spun at 500G for 4 minutes to pellet cells. The supernatant was removed, and the cells were processed using the RNeasy plus mini kit (Qiagen) following the manufacture protocol, including adding beta-mercaptoethanol to the lysis buffer. The cells were sent through the genomic DNA (gDNA) eliminator column supplied with the kit. One modification to the protocol was replacing the RW1 buffer with RWT buffer (Qiagen) for washing. The extracted RNA was eluted into RNAse-free water, and RNA integrity was checked using the Agilent bioanalyzer 2100 using the “high-sensitivity” setting. Only RNA with a RIN (RNA integrity number) > 8.5 was used for subsequent experiments. RNAs shorter than 100 nt were not efficiently recovered. RNA Seq libraries were prepared from enriched RNA (corresponding to ~30-50 µg of pre-enriched RNA) using the Illumina TruSeq Stranded mRNA Protocol, which included polyA+ selection. The prepared libraries were stranded and were quality-controlled by sequencing on the Illumina Miseq. Good libraries (> 80% unique reads) were sequenced on the Illumina Hiseq400 at ~35 million paired (2 x 75) reads per library. The correlation between biological replicates was high (between 0.96 and 1). For the MITO APEX Seq we also generated total RNA samples (by omitting the polyA+ selection step in the TruSeq protocol), as well as a polyA+ selected technical replicate for 1 of the labeled samples.