title: GSM7699258: 20220307_M41_WT_13Gy_FLASH_5M_2626; Mus musculus; RNA-Seq
library_construction_protocol: The trachea of the mouse was perfused with 1.5 ml of 50 U/ml dispase using, followed by 0.5 ml of 1% agarose. Lungs were minced using a scalpel to create small pieces and added into 3 ml of 1× DPBS MgCl2+and CaCl2+. Then 320 µl of 25 U/ml elastase were added and the suspension was homogenized at 37 °C using orbital shaking. The suspension was treated with 1× DPBS containing 10% fetal bovine serum (FBS) and 20 µl of 0,5 M EDTA pH 8 and filtered through 100 µm nylon cell strainer. The cells were treated with 37.5 µl of 10 mg/ml DNase I on ice, filtered through a 40 μm nylon cell strainer, centrifuged, resuspended in red blood cell (RBC) lysis buffer and incubated at RT before inhibition of the lysis using PF10 supplementation. After centrifugation, the pellet was resuspended in 1 ml of 1× DPBS containing 0.02% bovine serum albumin (BSA). Concentration of the samples was adjusted to 1 million cells/ml in 1× DPBS containing 0,02% BSA. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beats into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.