Nascent Transcriptome Profiling by 4-SU-seq

Nascent RNA-seq was performed as previously described (Kerry et al. 2017 (link)), after 96 h RUNX1 (SEM) or KMT2A-AFF1 (RS4;11) KD. Briefly, 1 × 10⁸ SEM cells were treated with 500 μM 4-thiouridine (4-SU) for 1 h. Cells were lysed with TRIzol (Invitrogen), and total RNA was precipitated and DNase I-treated. 4-SU-incorporated RNA was purified by biotinylation and streptavidin bead pulldown. DNA libraries were prepared using the Ultra II Directional RNA library prep kit (NEB, E7765) and sequenced by paired-end sequencing using a NextSeq 500 (Illumina).

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Harman J.R., Thorne R., Jamilly M., Tapia M., Crump N.T., Rice S., Beveridge R., Morrissey E., de Bruijn M.F., Roberts I., Roy A., Fulga T.A, & Milne T.A. (2021). A KMT2A-AFF1 gene regulatory network highlights the role of core transcription factors and reveals the regulatory logic of key downstream target genes. Genome Research, 31(7), 1159-1173.

Publication 2021

TRIzol Ultra II Directional RNA library prep kit NextSeq 500

Biotinylation Cells Dnase i Kmt2a Library Rna seq Runx1 Streptavidin Thiouridine Trizol

Corresponding Organization :

Other organizations : University of Oxford, MRC Weatherall Institute of Molecular Medicine, John Radcliffe Hospital

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Variable analysis

independent variables

RUNX1 knockdown (KD) in SEM cells
KMT2A-AFF1 knockdown (KD) in RS4;11 cells

dependent variables

Nascent RNA-seq (transcriptional profile)

control variables

Duration of knockdown (96 h)
Cell lines used (SEM and RS4;11)
Concentration of 4-thiouridine (4-SU) used (500 μM)
Duration of 4-SU treatment (1 h)
RNA extraction method (TRIzol)
RNA purification method (biotinylation and streptavidin bead pulldown)
DNA library preparation kit (Ultra II Directional RNA library prep kit)
Sequencing platform (NextSeq 500)

controls

No positive control mentioned
No negative control mentioned

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