Metabolic incorporation of electron-rich ribonucleosides enhances APEX-seq for profiling spatially restricted nascent transcriptome

We present MERR APEX-seq, a method for newly transcribed RNAs subcellular profiling combined metabolic incorporation of electron-rich ribonucleosides, 6-thioguanosine and 4-thiouridine, with the peroxidase-mediated RNA labeling method, APEX-seq. MERR APEX-seq offers both high spatial specificity and high coverage in the mitochondrial matrix and at the endoplasmic reticulum membrane. Application of MERR APEX-seq at nuclear lamina of human cells reveals that the mRNA components tend to encode for transcripts processing related proteins. MERR APEX-seq with high spatial specificity and high coverage could be widely used to expand our knowledge of RNA localization and function at subcellular compartments. MERR APEX-seq libraries were generated for the following constructs with APEX2: (1) MITO (mitochondrial matrix), (2) ERM (endoplasmic reticulum membrane), (3) NES (cytosol) and (4) LMNB1 (nuclear lamina). 16 libraries were prepared for MITO before (INPUT) and after (ENRICH) enrichment (2 biological replicates for labeled targets of s6G-APEX-labeling, s4U-APEX-labeling and APEX labeling each and 2 biological replicates for unlabeled control of no metabolism and no probe), 8 libraries were prepared for ERM before (INPUT) and after (ENRICH) enrichment (4 biological replicates for labeled targets of s6G-APEX-labeling), 4 libraries were prepared for NES before (INPUT) and after (ENRICH) enrichment (2 biological replicates for labeled targets of s6G-APEX-labeling) and 12 libraries were prepared for LMNB1 before (INPUT) and after (ENRICH) enrichment (2 biological replicates for labeled targets of s6G-APEX-labeling and APEX labeling each and 2 biological replicates for unlabeled control).