We prepared indexed paired-end libraries for Illumina sequencing as described (J. Z. Levin et al., Nature Methods 7, 709 (2010)., with the following modifications. First, we used a different indexing adaptor (containing an 8-base barcode) for each library. Second, we size-selected the ligation product by using two rounds of 0.7 volume of AMPure XP SPRI bead cleanup with the first round starting volume at 100   l. Third, we performed PCR with Phusion High-Fidelity DNA polymerase with GC buffer and 2 M betaine. Fourth, we used 55  C as the annealing temperature in PCR with the universal indexing primers (forward primer 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC, reverse primer 5'-CAAGCAGAAGACGGCATACGAGAT). Fifth, we performed 12 cycles of PCR. Sixth, we removed PCR primers using two rounds of 1.0 volume of AMPure beads.

We created a Bowtie index based on the UCSC knownGene (8) transcriptome, and aligned paired-end reads directly to this index using Bowtie v 0.12.7 with command line options -q --phred33-quals -n 2 -e 99999999 -l 25 -I 1 -X 1000 -a -m 200.

Next, we ran RSEM v1.11 with default parameters on these alignments to estimate expression levels. RSEM’s gene level expression estimates (tau) were multiplied by 1,000,000 to obtain transcript per million (TPM) estimates for each gene.

Supplementary_files_format_and_content: File allGenesTPM.txt represents a matrix of gene expression estimates across all non-MolecularBarcode samples. File umbExp.txt represents a matrix of gene expression estimates across all MolecularBarcode samples.  Linked as supplementary files on Series record.