Dataset Information

RNA pull down experiment for vA-202 (ANAPC1P2) lncRNA

ABSTRACT: This study aimed to identify protein interaction partners of the lncRNA vA-202 using an RNA pull-down approach followed by mass spectrometry. Biotinylated vA-202 RNA was generated by in vitro transcription incorporating biotin-14-CTP. For each pull-down reaction, 400 µg of whole-cell lysate (in a total volume of 100 µL) was incubated with 1 µg of biotinylated RNA in the presence of protease inhibitor cocktail (PIC), RNaseOUT, and 2× TENT buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 0.5% Tween-20), supplemented with IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol). The mixture was rotated at 4 °C for 2 hours. In parallel, streptavidin magnetic beads were washed three times with PBST (PBS + 0.1% Tween-20) and maintained on ice. The RNA-protein complexes were captured by adding the reaction mixture to the pre-washed magnetic beads and incubated for an additional 2 hours at 4 °C. Beads were then washed five times with PBST and twice with 100 mM ammonium bicarbonate. Bound proteins were eluted by incubating the beads in 0.1% RapiGest supplemented with 5 mM DTT at 60 °C for 30 minutes. The eluates were collected for downstream proteomic processing and LC-MS/MS analysis. Two controls were included: (i) a pull-down using an unrelated RNA (p-XEF) and (ii) a beads-only control (no RNA). All pull-down experiments were performed in triplicate.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Amruta Jadhav

LAB HEAD: Dr. Sharmila A. Bapat

PROVIDER: PXD070150 | Pride | 2026-05-25

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	BAPAT_FLRA_SET3_221021_1.raw	Raw
	BAPAT_FL_RA_SET1_20082021.raw	Raw
	BAPAT_FL_RA_SET2_221021_1.raw	Raw
	BAPAT_ONLYBEADS_SET1_021121.raw	Raw
	BAPAT_ONLYBEADS_SET2_221021.raw	Raw

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Publications

Emergence and evolution of chimeric pseudogenes (φgenes) contribute to genetic and functional diversity of the human genome.

Jadhav Amruta R AR Sahoo Sushmita S Kuber Yash L YL Hatwar Krutika K Varanwal Avinash A Tanwar Khushi K Palwankar Shubhankar S Chatrasal Gauri G Mali Avinash M AM Bapat Sharmila A SA

Nucleic acids research 20260401 8

Genome evolution through rearrangements and selection drives the emergence of new genes, gene families, and species. Although pseudogenes (φgenes) are often regarded as "dead on arrival" due to disruptive mutations or loss of regulatory elements, our earlier work revealed that several φgenes remain transcriptionally active, challenging this assumption. Here, we show that segmental duplications at proximal loci can generate chimeric φgenes comprising contiguous sequences from two or more genes, o ...[more]

PMID: 42049239

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