Polysome associated transcriptome in nonactivated and activated human CD8+ T cells
To capture the rapid changes of gene expression and translation upon T cell activation, we measured the transcriptome of total cell lysates and matched polysomal fractionation from non-activated primary human CD8+ effector T cells, and from CD8+ T cells that were reactivated for 2h with PMA-ionomycin. For polysome fractionation, we isolated RNA from 15 sucrose fractions (17-50% sucrose gradient), which based on 18S/28S ribosomal subunit distribution we pooled into ribosome-free, monosome-bound, polysome light and polysome heavy-bound RNA. Fractions were then purified and concentrated with RNA Clean & Concentrator-5 kit (Zymo Research). RNA for the whole cell transcriptome analysis was isolated with Quick-RNA Microprep kit (Zymo Research). RNA-sequencing analysis RNA-sequencing reads were quasi-mapped on the human transcriptome GRCh38 from Gencode (version 38, October 2021) using Salmon tool version 1.5.2. An average of 33×1e6 reads for each sample was mapped to the transcriptome.
To capture the rapid changes of gene expression and translation upon T cell activation, we measured the transcriptome of total cell lysates and matched polysomal fractionation from non-activated primary human CD8+ effector T cells, and from CD8+ T cells that were reactivated for 2h with PMA-ionomycin. For polysome fractionation, we isolated RNA from 15 sucrose fractions (17-50% sucrose gradient), which based on 18S/28S ribosomal subunit distribution we pooled into ribosome-free, monosome-bound, polysome light and polysome heavy-bound RNA. Fractions were then purified and concentrated with RNA Clean & Concentrator-5 kit (Zymo Research). RNA for the whole cell transcriptome analysis was isolated with Quick-RNA Microprep kit (Zymo Research). RNA-sequencing analysis RNA-sequencing reads were quasi-mapped on the human transcriptome GRCh38 from Gencode (version 38, October 2021) using Salmon tool version 1.5.2. An average of 33×1e6 reads for each sample was mapped to the transcriptome.
Sanquin