Single cell preparation for sequencing

Naïve and tumor-bearing liver tissues were processed with a consistent protocol as described previously [1] with an enzymatic collagenase/DNase treatment time of 13 min and PharMLyse (BD Biosciences) treatment of 5 min (tumor) and 10 min (liver, mix). HT-29 tumor single cell suspensions underwent a tumor cell depletion step as previously described [1].

Our single cell data were derived from multiple 10× Genomics sequencing runs. To benchmark batch effect in our data, each sample type (i.e. cells from naïve liver, tumor, adjacent normal or a mix type under certain genetic background) was subjected to multiple 10× Genomics runs spread across different days. Samples representing various samples types were collected on the same day and subsequently processed for sequencing. For the same sample type, batch effect was barely observed in cell clustering result.