title: Illumina HiSeq 4000 sequencing; Single cell sequencing of colorectal tumors and adjacent non-malignant colon tissue
design: Single cell sequencing of colorectal tumors and adjacent non-malignant colon tissue
library_construction_protocol: Following surgical resection, samples from the tumor (core and border regions for each tumor) and adjacent non-malignant tissue from the same resection specimen (maximal distance from the tumor is 5 cm) were taken from the pathologist. Samples were rinsed with PBS, and each sample was subsequently minced into smaller pieces of less than 1 mm3 on ice, and transferred to 10 mL digestion medium (DMEM [Gibco] with 2mg/ml of Collagenase P [Roche] and 0.2mg/ml of DNAse I [Roche]).  Samples were incubated for 15 minutes at 37°C, with manual shaking every 5 minutes. Samples were then vortexed for 10 seconds, and pipetted up and down for 1 minute using pipettes of descending sizes (25 mL, 10 mL and 5 mL). Next, 30 mL ice-cold phosphate-buffered saline, pH 7.4 (PBS [ThermoFisher Scientific]) was added, samples were filtered using a 40µm nylon mesh (ThermoFisher Scientific). Following centrifugation at 300 G at 4°C for 5 minutes, the supernatant was decanted and discarded, and the cell pellet was resuspended in 1 mL red blood cell lysis buffer (Sigma) and transferred to a 2 mL DNA low bind tube. Following a 5-minute incubation at room temperature, samples were centrifuged (150 G, 4°C, 5 min) using a swing-out rotor. Samples were next resuspended in 1 mL PBS containing 8 µL UltraPure bovine serum albumin (50 mg/mL, AM2616 [ThermoFisher Scientific]) and filtered over Scienceware Flowmi 40 µm cell strainers (VWR) using wide bore 1 mL low retention filter tips (Mettler-Toledo).  10 µL (9 µL of cell suspension plus 1 µL AOPI) of this cell suspension was next counted using an automated cell counter (Luna) to determine the concentration of live cells. Single cell suspensions were converted to barcoded 10x libraries by using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10X Genomics; Pleasanton, California, USA), aiming for an estimated 7,000 cells per library. Samples were processed using kits pertaining to V2 barcoding chemistry of 10X Genomics.