title: GSM6423830: CD45+ cells from Lewis lung carcinoma tumour from F11rfl/fl LysM-Cre- (myeloid JAM-A wild type) mice; Mus musculus; RNA-Seq
library_construction_protocol: Sample preparation for sort: To limit dissociation-induced gene expression, tumors were dissociated in the presence of actinomycin D (ActD), following a protocol described before (17). The concentration of ActD varied as follows: 30 μM during cutting, 15 μM during enzymatic digestion and 3 μM for all remaining steps. After tissue dissociation and antibody staining as described above, 45 000 7-AAD-CD45+ cells pooled from three size-matched tumors per genotype were sorted into RPMI with 10% (v/v) FCS, 300 μg/ml L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1% (v/v) MEM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 0.02 mM 2-mercaptoethanol (Sigma) and 3 μM ActD.   Post-sort sample preparation: Sorted cells were centrifuged for 6 min at 4°C at 400 g and resuspended in PBS with 0.04% bovine serum albumin in a final concentration of 1000 cells/µl. Library preparation: Single-cell suspensions were loaded on a GemCode Single Cell Instrument (10x Genomics) to generate single-cell gel beads-in-emulsion (GEM). GEMs and scRNA-Seq libraries were prepared using the GemCode Single Cell 3' Gel Bead and Library kit, version 3.1 (10x Genomics, No. 1000121) and the Chromium i7 Multiplex Kit (10x Genomics, No. 1000213) according to the manufacturer's instructions. Briefly, GEM reverse-transcription incubation was performed in a 96-deep-well reaction module at 53°C for 45 min, 85°C for 5 min and ending at 4°C. Next, GEMs were broken and complementary DNA (cDNA) was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, No. 37002D) and SPRIselect Reagent Kit (Beckman Coulter, No. B23318). Full-length, barcoded cDNA was PCR amplified with a 96-deep-well reaction module at 98°C for 3 min, fourteen cycles at 98°C for 15 s, 67°C for 20 s and 72°C for 1 min, one cycle at 72°C for 1 min and ending at 4°C.   Gene expression library construction to generate Illumina-ready sequencing libraries was performed after cleanup with the SPRIselect Reagent Kit and enzymatic fragmentation, by adding R1 (read 1 primer), P5, P7, i7 sample index and R2 (read 2 primer sequence) via end-repair, A-tailing, adapter ligation, post-ligation SPRIselect cleanup/size selection and sample index PCR. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent). Sequencing libraries were loaded on an Illumina NovaSeq 6000 flow cell with sequencing settings according to the recommendations of 10x Genomics (read 1: 26 cycles; read 2: 98 cycles; index i7: eight cycles; index i5: no cycles).