Bulk RNA-seq of Sorted or Cultured Cells

Bulk RNA-seq was performed using Smart-Seq2^{50 (link)} with minor modifications, as described in a previous study^{13 (link)}. Briefly, 5,000 sorted or cultured cells were resuspended in 15 μl of TCL (Qiagen), and their RNA was purified by a 2.2x SPRI cleanup with RNAClean XP magnetic beads (Agencourt). After reverse transcription, amplification and cleanup, libraries were quantified using a Qubit fluorometer (Invitrogen), and their size distributions were determined using an Agilent Bioanalyzer 2100. Amplicon concentrations to 0.1 ng/ml and sequencing libraries were constructed using a Nextera XT DNA Library Prep Kit (Illumina), following the manufacturer’s protocol. All RNA-seq libraries were sequenced with 38 × 38 paired-end reads using a NextSeq (Illumina). RNA-seq libraries were sequenced to a depth of >2 million reads per sample. STAR was used to align sequencing reads to the UCSC hg19 transcriptome and RSEM was used to generate an expression matrix for all samples. Both raw count and transcripts per million data were analyzed using edgeR and custom python scripts. The list of identified receptor-ligand pairs were obtained from a previous publication^{51 (link)}.