GRO-seq for Linear RAG Scanning Mediates Editing of Igκ Variable Region Repertoires
GRO-seq for Linear RAG Scanning Mediates Editing of Igκ Variable Region Repertoires
Briefly, 10 million cells were permeabilized with permeabilization buffer (10 mM Tris-HCl, pH 7.4, 300 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, one tablet of protease inhibitors per 50 ml and 4 units Rnase inhibitor per ml) and resuspended in 100 ul storage buffer (10 mM Tris-HCl, pHHH 8.0, 25% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM EDTA and 5 mM DTT). Permeabilized cells were subjected to nuclear run-on (2xRun-on mix: 5 mM Tris-Cl, PH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.5 mM BrUTP, 10 units of RNase inhibitor, 1% sarkosyl) at 37o for 5 min, followed by Trizol-based RNA extraction. v-Abl cell lines were cultured in RPMI1640 media with 15% FBS G1-arrest was performed by treating v-Abl cell lines with 3uM STI for 4 days Briefly, 10 million cells were permeabilized with permeabilization buffer (10 mM Tris-HCl, pH 7.4, 300 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, one tablet of protease inhibitors per 50 ml and 4 units Rnase inhibitor per ml) and resuspended in 100 ul storage buffer (10 mM Tris-HCl, pHHH 8.0, 25% (vol/vol) glycerol, 5 mM MgCl2, 0.1 mM EDTA and 5 mM DTT). Permeabilized cells were subjected to nuclear run-on (2xRun-on mix: 5 mM Tris-Cl, PH 8.0, 2.5 mM MgCl2, 0.5 mM DTT, 150 mM KCl, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.5 mM BrUTP, 10 units of RNase inhibitor, 1% sarkosyl) at 37o for 5 min, followed by Trizol-based RNA extraction. Illumina adapters were ligated to RNA followed by cDNA generation with reverse transcriptase.  Libraries were PCR-amplified with Illumina primers and subjected to sequencing.