title: GSM7250145: 3P-Seq in undifferentiated SH-SY5Y cells; Homo sapiens; OTHER
library_construction_protocol: RNA was harvested using Trizol. 75 ug total RNA was used for input in library preparations RNA libraries for 3P-seq were prepared as described in Jan et al., 2011 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057491/) with the following exceptions: After elution of the mRNA 3′ ends from oligo-d(T) beads, a mixture of two pre-adenylated 3′ DNA adapters (AppNN NGT ANN NTA GNN NGA TCG TCG GAC TGT AGA ACT CTG AAC /3AmMO/, AppNN NAC TNN NGA TNN NGA TCG TCG GAC TGT AGA ACT CTG AAC /3AmMO/), adenylated as described in Hildebrand et al., 2022 (https://pubmed.ncbi.nlm.nih.gov/37059467/) were ligated to these eluted mRNAs using Rnl2KQ. The ligation products were gel purified and phosphorylated as described in Jan et al., 2011 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057491/)  and a 5′ RNA adapter (rCrCrU rUrGrG rCrArC rCrCrG rArGrA rArUrU rCrCrA rNrNrN rCrUrA rNrNrN rGrArC rNrNrN) was ligated to the 5′ RNA ends using Rnl1. This ligation product was then reverse transcribed using Superscript 2  with the primer sequence: GTT CAG AGT TCT ACA GTC CGA CGA TC. The RT product was then gel purified and the final libraries were amplified using AccuPrime pfx DNA polymerase and the following primers: Forward Primer: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA and Reverse Primer: CAA GCA GAA GAC GGC ATA CGA GAT CGT GAT GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA. The final amplified libraries were gel extracted, quantified, and sequenced on a NextSeq 550 (Illumina) for 79 cycles.