cdna library construction protocols  (Oxford Nanopore)

Journal: Protein & Cell

Article Title: A molecular brake that modulates spliceosome pausing at detained introns contributes to neurodegeneration

doi: 10.1093/procel/pwac008

Figure Lengend Snippet: DI splicing is likely paused at B act . (A) Potential SNIP1 interacting proteins identified by Flag-IP coupled with mass spectrometry (co-IP/MS) in N2a cells expressing Flag-SNIP1. Many of these protein partners are involved in pre-mRNA splicing and RNA metabolism (also see ). (B) The potential SNIP1 interacting partners are found in B act but not B*. Factors loaded into B* are labeled in gray, which were not hit by our co-IP/MS. (C and D) Interactions between SNIP1 and protein components found in B act component SF3a120 (C) but not those in B* component YJU2 (D) were validated in N2a cells expressing the indicated tagged proteins. (E) RIP was performed in N2a cells expressing Flag-SF3a120, Flag-YJU2, or EGFP. DIs were measured by RT-PCR using Smart-amplified cDNA. (F–I) Flag-RIP-seq was performed in N2a cells expressing Flag-SF3a120 (F) and Flag-YJU2 (G). N2a cells expressing EGFP, a negative control for our Flag-RIP-seq. Two representative SF3a120-bound DIs visualized by IGV (H and I). See also , .

Article Snippet: Total RNA (1 μg) was used for cDNA library construction, following the protocols suggested by Oxford Nanopore Technologies (ONT).

Techniques: Mass Spectrometry, Co-Immunoprecipitation Assay, Expressing, Labeling, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control