title: GSM9648638: RNA-seq for GS59; Homo sapiens; OTHER
library_construction_protocol: HeLa genomic DNA purchased from NEB, no. N4006S Experimental process for ribozyme identification. HeLa genomic DNA libraries were constructed using the tagmentation method, which simultaneously fragments genomic DNA (NEB, no. N4006S) and incorporates adapter sequences via the Tn5 transposase. The process was performed using the Nextera XT DNA Library Preparation Kit (Illumina, no. FC-131-1024), following the manufacturer′s guidelines. PCR amplification of the tagmentation product was carried out using primers T7p-s7 and T7t-s5. These primers were designed to anneal to the Nextera s7 and s5 adapter regions, respectively, and incorporated the T7 promoter sequence at the 5′ ends of the amplicons.  Sequences of all oligonucleotides used in this study are listed in Supplementary Table 1. Separation of the DNA library fragments (500-1000 bp) was achieved by agarose gel electrophoresis and subsequent gel purification. Purified DNA library was then used as the template for in vitro transcription by incubation with 100 U of T7 RNA Polymerase (NEB, no. M0251L) in 1×T7 RNA polymerase buffer (40 mM Tris-HCl pH 7.9, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine) with 2 mM NTP mix (NEB, no. N0466S) and 20 U Murine RNase Inhibitor (NEB, no. M0314S) in a 30 µl reaction volume for 5 h at 37 °C. DNA templates were removed by incubation with 2 U of DNase I (NEB, no. M0303S) and 1× DNase I reaction buffer in a 100 µl volume at 37 °C for 15 min, followed by purification with  RNA Clean & Concentrator-5 kit (Zymo Research, no. R1016). 2.5 µg purified RNA were incubated with 100 pmol linker (rM13F_3desBio for 3P-seq, rM13R_5desBio_3P for 5OH-seq) in 1× RtcB reaction buffer with 22.5 pmol RtcB ligase (NEB, no. M0458S), 0.1 mM GTP and 1 mM MnCl2 in 30 µl at 37 °C for 1 h. Ligation products were purified with Sera-Mag Streptavidin-Coated Magnetic Beads (Cytiva, no. 30152103010150) and eluted in 20 µl TNB buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 4 mM biotin). Before reverse transcription, ligation products were exchanged into H2O by column purification. Ligation products were reversed transcribed using primer (RT1_m13f_adp1 for 3P-seq, RT1_Tn5ME_adp1 for 5OH-seq) and the ProtoScript II Reverse Transcriptase (NEB, no. M0368S). Ligation product was first denatured at 65 °C for 5 min in a 19.5 µl volume containing 1 µl of 100 µM primer and 1.5 µl of 10 mM dNTP mix, followed by rapid snap-cooling on ice for 2 min. Then, 6 µl of 5 × ProtoScript II Reverse Transcriptase buffer, 3 µl of 0.1 M DTT, 0.5 µl of Murine RNase Inhibitor (NEB, no. M0314S) and 1 µl of ProtoScript II Reverse Transcriptase were added and incubated at 42 °C for 1 h and then 65 °C for 20 minutes to inactivate the enzyme, followed by enrichment with Sera-Mag Streptavidin-Coated Magnetic Beads (Cytiva, no. 30152103010150) and clean-up by using RNA Clean & Concentrator-5 kit (Zymo Research, no. R1016). Purified cDNA was amplified by touch-up PCR using primers (P7R2_Tn5ME/P5R1_adp1 for 3P-seq, P7R2_m13r/P5R1_adp1 for 5OH-seq) with Q5 Hot Start High-Fidelity DNA Polymerase (NEB, no. M0493S) to construct the high-throughput sequencing library. The PCR products were purified with a homemade magnetic bead mix and sequenced on an Illumina HiSeq X sequencer with 20% PhiX control by Novogene Technology Co., Ltd.  Mutational profiling of four identified ribozymes. Shorter versions of the identified ribozymes were defined by sequential truncation of terminal regions that did not significantly reduce cleavage activity. These truncated sequences were used for subsequent mutational‑scanning experiments. A random mutagenesis library was generated using a commercial Random Mutagenesis Kit (Biosharp, no. BL1668A). Barcode sequences were appended to the library in three PCR cycles. Approximately 10⁶ barcoded molecules were used as template to amplify the final mutant DNA library. The final mutant DNA library served as template for constructing the DNA‑seq library. The barcoded DNA library was transcribed with T7 RNA Polymerase (Beyotime, cat. no. R7012L) for 4 h at 37 °C. After transcription, the DNA template was removed by treatment with DNase I (New England Biolabs, no. M0303S). The purified RNA was incubated in 1× TKMS buffer (50 mM Tris‑HCl pH 8.0, 25 mM KCl, 10 mM MgCl₂, 2 mM spermidine) at 37 °C for 2 h to allow ribozyme cleavage, followed by purification with the RNA Clean & Concentrator‑5 kit (Zymo Research, cat. no. R1016). For reverse transcription, ~50 pmol of RNA was mixed with 50 pmol of RT primer and 200 pmol of template‑switch oligo (TSO), and reverse‑transcribed using M‑MLV (H‑) Reverse Transcriptase (Vazyme, no. R021) at 42 °C for 1 h. The purified cDNA was used as the template for NGS library amplification. PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs, no. M0491L) for 12 cycles. The PCR products were purified with a homemade magnetic bead mix and sequenced on a Salus EVO sequencer by Shenzhen Salus BioMed Co., Ltd as the RNA-seq library.