ena-STUDY-INSTITUTE OF BIOCHEMISTRY AND BIOPHYSICS, POLISH ACADEMY OF SCIENCES-18-06-2019-10:50:17:773-31
B cell humoral response and differentiation is regulated by non-canonical poly(A) polymerase TENT5C.
The goal of this study was to assess changes in the activated B cells transcriptome upon TENT5C knockout in mice. Primary B cells were isolated from either wild-type or TENT5C KO mice then activated in vitro with 20 ng/ml IL-4 (Peprotech) and 20 ??g/ml LPS (Santa Cruz) for 7 days.  RNA isolation was performed with Trizol reagent (Invitrogen, 15596) according to the manufacturer's instructions, followed by mRNA enrichment with GST-eIF4E-K119A protein and ribodepletion using a Ribo-Zero Kit (Epicentre, RZG1224), according to the manufacturer's recommendations.  Nanopore direct-RNA libraries were prepared from 500 ng of cap-enriched, rRNA-depleted mRNA with Direct RNA Sequencing Kit (ONT, SQK-RNA001). Sequencing was performed with MinION device (Flow cell type R9.4.1; RevC) and basecalled using Guppy 2.2.2 (replicates 1 and 2) or Guppy 3.3.0 (replicates 3) (Oxford Nanopore Technologies).
The goal of this study was to assess changes in the activated B cells transcriptome upon TENT5C knockout in mice. Primary B cells were isolated from either wild-type or TENT5C KO mice then activated in vitro with 20 ng/ml IL-4 (Peprotech) and 20 ??g/ml LPS (Santa Cruz) for 7 days.  RNA isolation was performed with Trizol reagent (Invitrogen, 15596) according to the manufacturer's instructions, followed by mRNA enrichment with GST-eIF4E-K119A protein and ribodepletion using a Ribo-Zero Kit (Epicentre, RZG1224), according to the manufacturer's recommendations.  Nanopore direct-RNA libraries were prepared from 500 ng of cap-enriched, rRNA-depleted mRNA with Direct RNA Sequencing Kit (ONT, SQK-RNA001). Sequencing was performed with MinION device (Flow cell type R9.4.1; RevC) and basecalled using Guppy 2.2.2 (replicates 1 and 2) or Guppy 3.3.0 (replicates 3) (Oxford Nanopore Technologies).