title: RNA-seq of esophageal tumor specimen
design: Tissue was retrieved via endoscopic biopsy at several stages throughout the patients treatment pathway including treatment nave, mid-treatment (after two cycles of aDCF) and post-treatment pre-resection samples. Using high-definition white light endoscopy to ensure the efficacy of sample collection, multiple biopsies were taken from the suspected lesion with an additional sample taken from the same location. The former was placed into a cold medium of RPMI (Invitrogen), Primocin (Invivogen) and gentamycin (Invitrogen). To achieve single cell disassociation, specimens were dissected minced and digested using 5 mL of Advanced DMEM/F12 containing 10 mg Collagenase Type 3 (Worthington) and 500 U Hyaluronidase (Sigma) in a Ctube (Miltenyi) using the gentleMACS Octo Dissociator (Miltenyi) and resuspended in PBS and 1MM of DDT. The suspension was passed through a 100um cell strainer (Fisher) and centrifuged (500xg at 5 minutes, 4oC). The cells were resuspended in 0.25% Trypsin-EDTA (Invitrogen) and subsequently incubated for 5 minutes at 37oC. The trypsin was then inactivated by adding 10% fetal bovine serum. After being re-centrifuged (500xg, 5 minutes, 4oC) the cell pellet was resuspended in 2.5U Dispase/10ug DNAse buffer and incubated for 5 minutes at 37oC which was then inactivated by adding PBS. The homogenate was then passed though a 40 uM strainer (Fisher) prior to being centrifuged again (500xg, 5 minutes, 4oC). Using ACK Lysing Buffer (Gibco) for 5 minutes at room temperate, red cells were lysed. After adding PBS, and being centrifuged again (500xg, 5 minutes, 4oC), the cell pellet was washed twice with 2% fetal bovine serum in PBS. The substrate was then processed via the 10x Genomics platform. The LightCycler 480 Real Time PCR instrument (Roche) and the KAPA library quantification kid (Roche) were used to quantify cellular libraries with triplicate measurements. These quantification values were used for MGI library conversion and for Illumina sequencing normalization. After 10x library construction, the MGIEasy Universal Library Conversion kit was used ensure all outputs were compatible with MGI sequencers. This conversion circularizes the library by rolling circle amplification that creates a long DNA strand which individually fold into a tight nanoball where each library fragment results in a single DNA nanoball. The DNA nanoballs were quantified with Qubit ssDNA HS assay kit (ThermoFisher), normalized and loaded into the sequencing flowcells using an auto-loading methods (MGI-DL-200R autoloader). The flowcells have a functional surface that captures and immobilizes the DNA nanoballs in a grid like pattern. Two libraries were typically loaded per lane for the single cell RNA sequencing using the DNBSEQ-G400RS PE100 MGI kit with App-A primers on a DNBSEQ-G400 MGI sequencer. Single cell RNA libraries were sequenced with 28 cycles for Read 1, 150 cycles for Read 2 and 8 cycles for the i7 index. Color balancing was achieved with color-balanced single index adapters (10x Genomics) were used for libraries sequenced on MGI. These MGI runs were demultiplexed by fastq-multx In both instances we used a mismatch of 1. After polyA-trimming via cutadapt (v3.2)86 , reads were pseudo-aligned to the GRCh38 reference transcriptome (ENSEMBL release 96) with kallisto (v0.46.2)87 using the default kmer size of 31. The pseudo-aligned reads were processed into a cell-by-gene count matrix using bustools (0.40.0)