SNAI2 ChIP-seq Protocol for Gene Regulation

ChIP-seq data used, was published previously (28 (link)) and performed as follows. 1% Formaldehyde-fixed chromatin from RD and SMS-CTR cells were sheared to 200–700 bp with Active Motif EpiShear Sonicator. Chromatin-IP with SNAI2 Ab (CST, Catalogue # 9585) was performed O/N, using ChIP-IT High Sensitivity kit (Active Motif). Drosophila chromatin (Active Motif, Catalogue #53083) and H2Av ab (Active Motif, Catalogue #61686) was used for spike-in normalization across samples. ChIP-seq libraries were prepared using Illumina TruSeq ChIP Library Prep Kit and sequenced on NextSeq500. Reads were mapped to reference genome (version hg19) using BWA. High-confidence ChIP-seq peaks were called by MACS2.1. Raw sequencing data and processed files are available through GEO (GSE137168). For real-time qPCR immunoprecipitated DNA was amplified with BCL2L11 ChIP primers (SNAI2 binding peak 1 & 2) along with negative controls. All signals were normalized against input by the percentage input method. Significance was calculated by Unpaired t test.

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Wang L., Hensch N.R., Bondra K., Sreenivas P., Zhao X.R., Chen J., Campos R.M., Baxi K., Vaseva A.V., Sunkel B.D., Gryder B.E., Pomella S., Stanton B.Z., Zheng S., Chen E.Y., Rota R., Khan J., Houghton P.J, & Ignatius M.S. (2021). SNAI2-mediated repression of BIM protects rhabdomyosarcoma from ionizing radiation. Cancer research, 81(21), 5451-5463.

Publication 2021

EpiShear Sonicator ChIP-IT High Sensitivity kit Drosophila chromatin TruSeq ChIP Library Prep Kit

Cells Chip Chip seq Chromatin Drosophila Formaldehyde Genome Library Primers Sensitivity

Corresponding Organization :

Other organizations : The University of Texas Health Science Center at Houston, The University of Texas Health Science Center at San Antonio, Wenzhou Medical University, Nationwide Children's Hospital, Case Western Reserve University, Bambino Gesù Children's Hospital, The Ohio State University, University of Washington, Center for Cancer Research

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Variable analysis

independent variables

Formaldehyde-fixed chromatin from RD and SMS-CTR cells

dependent variables

SNAI2 binding sites identified by ChIP-seq
Expression of BCL2L11 gene measured by real-time qPCR

control variables

Chromatin sheared to 200–700 bp using Active Motif EpiShear Sonicator
Chromatin-IP with SNAI2 antibody (CST, Catalogue # 9585) performed O/N using ChIP-IT High Sensitivity kit (Active Motif)
Drosophila chromatin (Active Motif, Catalogue #53083) and H2Av antibody (Active Motif, Catalogue #61686) used for spike-in normalization
ChIP-seq libraries prepared using Illumina TruSeq ChIP Library Prep Kit and sequenced on NextSeq500
Reads mapped to reference genome (version hg19) using BWA
High-confidence ChIP-seq peaks called by MACS2.1
Immunoprecipitated DNA amplified with BCL2L11 ChIP primers (SNAI2 binding peak 1 & 2) and negative controls
All signals normalized against input by the percentage input method

positive controls

Drosophila chromatin (Active Motif, Catalogue #53083) and H2Av antibody (Active Motif, Catalogue #61686) used for spike-in normalization

negative controls

Negative controls used for real-time qPCR of immunoprecipitated DNA

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