title: Gene expression library for sample Seq098and099_Lib5
design: Biopsies were minced into 2mm^2 chunks using a sterile razor in a tissue culture dish containing 5mL RPMI with 5% FBS and 50uL Penicillin-Streptomycin (P/S, Thermo Fisher cat. 15140122). Tissue fragments were then placed in a dissociation enzyme mix of 10mL of RPMI with 2mg/mL Collagenase A (Roche 10103586001), 0.1mg/mL DNAse (Roche), and 10uL P/S. The tissue was dissociated for 15 minutes at 37C under rotation at a 45-degree angle. The supernatant containing dissociated cells was removed, leaving behind solid settled chunks, and set on ice. A fresh dissociation enzyme mix was added to the remaining tissue fragments, and a second 15-minute dissociation was completed. Supernatants from both rounds of dissociation were pooled and cells were pelleted at 400g for 5min at 4C in a swinging bucket centrifuge. Cells were washed with PBS and filtered through a 70 µm cell strainer. Cells were pelleted again and resuspended in 500uL of PBS supplemented with 0.1% BSA and stored on ice until used. The maximum time between dissociation and cell encapsulation was approximately 4 hours. inDrops libraries were generated by the Harvard Longwood Single-Cell Core. Cells were provided to the core in PBS with 0.1% BSA. Cells were run through a microfluidic chip with barcoded hydrogels, and the inDrops v3 protocol was applied to generate sequencing libraries with a target of 2,000 cells per library. inDrops libraries were sequenced by the MGH NextGen Sequencing Core on an Illumina HiSeq2500, in either High Output or Rapid mode, depending on the number of multiplexed libraries. Read lengths were R1:65, R2:8, R3:8, and R4:26. All libraries were sequenced to a target depth of 100,000 reads per cell.