Depletion of dMes-4/NSD and Hypb/Set2 in S2 cells

Exponentially growing S2 cells were depleted by dsRNAs against dMes-4/NSD or Hypb/Set2 compared with mock-depletions (dsRNAs against luciferase) as previously described (Lhoumaud et al, 2014 (link); Liang et al, 2014 (link)). Preparation of dsRNAs was done using the indicated oligos by T7-driven transcription (Fermentas TranscriptAidTM T7 High Yield Transcription Kit). Depletions were verified by quantitative RT-qPCR analysis using cDNAs prepared from control, dMes-4/NSD-, or Hypb/Set2-depleted cells, with the indicated oligos. Gene expression analyses by RNAseq were performed as previously described (Lhoumaud et al, 2014 (link); Liang et al, 2014 (link)) in cells depleted of dMes-4/NSD or Hypb/Set2 compared with control (GSE146992). For analysis of Pol II pausing, cells were treated with either flavopiridol (3055; Sigma-Aldrich) at 1 μM during 30 min, or DRB (D1916; Sigma-Aldrich) at 50 μM during 30 min, or DMSO control (23500-260; VWR).

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Depierre D., Perrois C., Schickele N., Lhoumaud P., Abdi-Galab M., Fosseprez O., Heurteau A., Margueron R, & Cuvier O. (2023). Chromatin in 3D distinguishes dMes-4/NSD and Hypb/dSet2 in protecting genes from H3K27me3 silencing. Life Science Alliance, 6(11), e202302038.

Publication 2023

TranscriptAidTM T7 High Yield Transcription Kit flavopiridol

Cdnas Cells Dmes Dmso Dsrnas Flavopiridol Gene expression analyses Luciferase Oligos Pol ii Transcription

Corresponding Organization :

Other organizations : Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, Université Toulouse III - Paul Sabatier, Centre National de la Recherche Scientifique, Université Paris Sciences et Lettres, Inserm, Institut Curie

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Variable analysis

independent variables

Depletion of dMes-4/NSD or Hypb/Set2 using dsRNAs
Treatment with flavopiridol, DRB, or DMSO control

dependent variables

Gene expression measured by RNA-seq
Pol II pausing

control variables

Growth of S2 cells (exponentially growing)
Preparation of dsRNAs using T7-driven transcription
Depletion verification by RT-qPCR
Previously described methods for RNA-seq analysis (Lhoumaud et al, 2014; Liang et al, 2014)

controls

Positive control: dsRNAs against luciferase (mock-depletion)
Negative control: DMSO treatment

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