GSM5325967: HEK_Hpos_ILMN; Homo sapiens; OTHER
Libraries from RPFs isolated from HEK293T cells were prepared using the SMARTer® smRNA-Seq Kit for Illumina  and sequenced with 50 cycles single-read on an Illumina NovaSeq 6000 sequencer. Illumina libraries for RPFs from mouse liver tissue were prepared according to Ingolia et al., 2012 and were sequenced with 50 cycles single-read on an Illumina HiSeq2500 sequencer. HEK298T cell lysates were obtained using a hypotonic lysis buffer. A total of 1.7 a.u. (A260 nm) were digested using . RPFs were generated by treating 1.7a.u. of lysate with 12.7 U of RNase I at room temperature for 45 min (as described in Clamer et al., 2018). ). Liver tissues were dissected immediately following sacrifice and pulverized under liquid nitrogen using a pestle and a mortar and the lysates obtained according to Bernabò et al.2017. Lysates were treated with RNAse I and the 80S with RPFs were isolated using sucrose gradient separation. The RPFs were purified using acidic phenol/chloroform extraction followed by PAGE extraction. Libraries from RPFs isolated from HEK293T cells were prepared using the SMARTer® smRNA-Seq Kit for Illumina  and sequenced with 50 cycles single-read on an Illumina NovaSeq 6000 sequencer. Illumina libraries for RPFs from mouse liver tissue were prepared according to Ingolia et al., 2012 and were sequenced with 50 cycles single-read on an Illumina HiSeq2500 sequencer. Oxford nanopore libraries for RPFs from HEK293T and mouse liver were prepared according to circAID-p-seq protocol. Briefly, upon 5′ phosphorylation of RPFs, library preparation was performed starting from 10 pmol of ADR12 used for ligation, followed by RNA self circularization and RT-RCA. The final libraries were loaded on a R9.4 flow cell and sequenced with a MinION sequencer.