Protoscript 2 buffer

A total of 0.7 μl RT Primer (from a 100 μM stock) was added to the purified sample and incubated at 75°C for 3 min, 37°C for 10 min and 25°C for 10 min. A total of 4 μl of ProtoScript II Buffer, 1 μl dNTP mix (NEB), 0.2 μl 1 M MgCl₂, 2 μl DTT (NEB) and 2 μl of ProtoScript II were added and the mixture incubated at 42°C for 12 h with a 105°C heated lid. (Alternatively, for standard conditions [see section on 2′-5′ linkages], no MgCl₂ was added and the incubation was at 50°C for 1 h.) Ten microliter of water was added and the mixture was run through an Oligo Clean & Concentrator spin column, including the RNA degradation step as per the manufacturer's instructions. The cDNA was eluted in 30 μl of TE, pH 7 and stored at 4°C. The cDNA stock concentration was measured with a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific), and typically found to be in the fraction of a μg/μl range.

Total RNA was extracted from 50 million SHEP MYCN-ER cells spiked-in with 15% Drosophila S2 cells using TRIzol reagent (Thermo Fisher Scientific, #15596018). mRNA was purified from 1 μg of total RNA with the VAHTS mRNA capture beads (Vazyme, #N401-02) and fragmented to 200 to 500 bp size long in 2× ProtoScript II buffer (New England Biolabs, #M0368). First- and second-strand DNA synthesis and adaptor ligation were conducted using an New England Biolabs Next Ultra RNA library prep kit for Illumina. The resulting strand-specific RNA-seq libraries were subjected to 150-bp paired-end sequencing on a NovaSeq 6000. All raw fastq reads were aligned to the Drosophila genome (dm6) and human genome (hg38) by HISAT2 version 2.1.0, respectively. Raw read counts were normalized to RPM (reads per million) per sample and then displayed in the UCSC genome browser as bigWig-formatted coverage tracks. Read counts at indicated regions were calculated by HTSeq-count (version 0.12.3) and were provided to DEseq2 (version 1.32.0) for differential gene expression analysis. Human gene expression was normalized using Drosophila spiked-in genes. FDR < 0.05 and log₂FC >0.5 (or < −0.5) were used to identify differentially expressed genes.