Single-cell combinatorial indexing (‘sci-’) is a methodological framework that employs split-pool barcoding to uniquely label the nucleic acid contents of large numbers of single cells or nuclei. The sci-RNA-seq3 is an updated version of sci-RNA-seq. The major improvements are: (1) nuclei are extracted directly from fresh tissues without enzymatic treatment; (2) hairpin ligation for the third level indexing (barcoded Tn5 tagmentation was used in the previous version); (3) individually optimised enzymatic reactions; (4) FACS was replaced by dilution, and sonication and filtration steps were added to minimize aggregation. [ http://dx.doi.org/10.17504/protocols.io.9yih7ue ]