GSM9533538: 10X Fixed RNA-seq: 13 Lung samples (2 cell lines): CD45 negative (HDM, 2xNRG1, PBS)  CD45positive (3xHDM, 3xNRG1, 3xPBS); Mus musculus; RNA-Seq
Whole lungs were removed from selected mice and incubated in digest media for 45 min followed by the addition of EDTA (2 mM final concentration) for 15 min. Digest medium consisted of Dulbecco's modified Eagle's media supplemented with 5% fetal calf serum, sodium bicarbonate, penicillin/streptokinase, 10 mM HEPES, 5 U/ml Hyaluronidase, 50 u/ml DNAse and 40 U/mL Liberase . To make cell suspension lung tissue was disrupted using Gentle MACS dissociator (Milteney Biotec). The samples were filtered and the single cell suspension was treated with Red Blood Cell Lysing Buffer (Sigma-Aldrich). Cells were then stained with Alexa Fluor® 488 anti-mouse CD45.2 antibody clone 104  (cat# 109816, Biolegend) using standard techniques, and FACS was performed sorting for both CD45+ and CD45- populations. Samples were prepared for single-cell RNA sequencing  using Next GEM Single Cell Fixed RNA sample preparation kit (cat# 1000414) following the manufacturer's protocol (CG000782 Rev C, 10X Genomics). For long term storage fixed cells were spun down at 850 rcf, 5 min and 0.1 volume (10%) of the pre-warmed enhancer (10Xgenomics, #1000414) was added to fixed samples in quenching buffer and 50% glycerol (Millipore Sigma, #G5516-100ml). Pipette mixed and stored at (-80°C) for use within 3 months. By quantification of the total cell number of each sample 13  samples were qualified to proceed to do the preparation of scRNA-seq libraries using Chromium fixed RNA profiling reagent kits No. 1000497 and Dual Index kit TS set A, 96 rxn No. 1000251 according to the manufacturer's protocol (CG000527 Rev D, 10X Genomics) followed by sequencing using Novaseq6000 platform and Flow cell-type is SP100-cycle. Samples were prepared for single-cell RNA sequencing following the manufacturer's protocol (CG000782 Rev C, 10X Genomics). The fixed cell suspensions were then prepared for sequencing library generation using Chromium fixed RNA profiling reagent kits No. 1000497 and Dual Index kit TS set A, 96 rxn No. 1000251 according to the manufacturer's protocol (CG000527 Rev D, 10X Genomics) and Chromium Next GEM Chip Q Single Cell Kit, 16 rxns (PN-1000422).  The pooled barcode cDNA which contained the 10x Barcode was amplified for subsequent library construct. Library preparation type was 10x fixed RNA profiling for mouse specie. Illumina Sequencing platform was NovaSeq-SP full cell. During the library construction paired-end constructs, with index (i7) sequence [CAAGCGTTAG] and the index (i5) sequence [CGCATTCGCG]  for reverse complimentary strand, were added.