title: GSM6634342: Lung KP DT (Diphtheria Toxin 48hr) sample #D2, visium; Mus musculus; OTHER
library_name: GSM6634342
library_construction_protocol: For isolation of immune and stromal cells, lungs were perfused, placed into 5 ml Eppendorf tubes containing 400ml of cold serum-free RPMI and chopped with scissors (1-2 mm). Lung fragments were placed in 2-3 ml of pre-warmed digestion medium (RPMI 1640, 10 mM HEPES buffer, 1% penicillin–streptomycin, 1% L-glutamine, liberase (Sigma-Aldrich #05401020001) and 1 U/ml DNase I (Sigma-Aldrich #10104159001)) (2-3 ml) and incubated for 30 min at 37°C. After digestion supernatant was collected and cells were resuspended in ice-cold RPMI1640 containing 5% FCS (ThermoFisher #35010CV), 1mM HEPES (Corning #MT25060CI), 1% penicillin–streptomycin (Corning #MT30002CI) and 200mM L-glutamine (Corning #MT25005CI). After additional digestion for 1 hr of the remaining tissue, both digested cell fractions passed through a 100um strainer (Corning #07-201-432), washed and FACS sorted. Bulk RNA seq - Cell populations were sorted straight into Trizol (ThermoFisher #15596018), RNA was precipitated with isopropanol and linear acrylamide, washed with 75% ethanol, and resuspended in RNase-free water. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 0.4-2.0ng total RNA with RNA integrity numbers ranging from 1.0 to 9.9 underwent amplification using the SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech #63488), with 12 cycles of amplification. Subsequently, 1.5-10ng of amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems #KK8504) using 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 or HiSeq 2500 in Rapid Mode in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit or HiSeq Rapid SBS Kit v2 (Illumina).  An average of 32 million paired reads were generated per sample and the percent of mRNA bases per sample ranged from 62% to 88%. scRNAseq- Single-cell RNA-Seq was performed on FACS sorted mouse lung KP cells or patient LuAd samples, on the Chromium instrument (10X genomics) following the user guide manual (CG00052 Rev E) for 3′ v2 and v3 as previously described46.  Briefly, sorted cells were washed once with PBS containing 0.04% bovine serum albumin (BSA) and resuspended in PBS containing 0.04% BSA to a final concentration of 700–1,200 cells per μl. Viability of cells was confirmed to be above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Then samples were encapsulated in microfluidic droplets at a dilution of ∼70 cells/μl (doublet rate ∼3.9%.). Encapsulated cells were subjected to reverse transcription (RT) reaction at 53°C for 60 min. After RT, the emulsion droplets were broken and barcoded-cDNA was purified with DynaBeads MyOne SILANE, followed by 14-cycles of PCR-amplification (98°C for 180 s; [98°C for 15 s, 67°C for 20 s, 72°C for 60 s] x 12-cycles; 72°C for 60 s). 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit and purified with SPRI beads to obtain an average fragment size of 600 bp. Next, the DNA library was ligated to the sequencing adaptor followed by indexing PCR (98°C for 45 s; [98°C for 20 s, 54°C for 30 s, 72°C for 20 s] x 10 cycles; 72°C for 60 s). An average of 5,000 cells were targeted for each tumor sample. The resulting DNA library was double-size purified (0.6-0.8X) with SPRI beads and sequenced on an Illumina NovaSeq platform (R1 – 26 cycles (KP) 28 cycles (LuAd), i7 – 8 cycles, R2 – 96 cycles (KP), 90 cycles (LuAd)) resulting in 184.5-186.1 million reads per sample (average reads per single-cell being 42,000 and average reads per transcript 4.40-7.14) (KP)