GSM6649522: P3504_1142; Mus musculus; RNA-Seq
For full-length (Smart-Seq2) library preparation (Picelli et al., 2014), the left lungs were used for enzymatic digestion and right lungs were treated as described above for histological analysis. Briefly, we cut the lungs in small pieces using a razor blade and digested with elastase (Worthington, LS002292) and DNase I 0.5 mg/ml (Sigma-Aldrich, DN25) in HBSS (Gibco, 14175), at 37oC for 1 hour with rotation. Equal volume of HBSS++ [HBSS (Gibco, 14175), supplemented with 2% 0.2µm filtered FCS (Gibco, 10500), 0.1 M HEPES (Sigma-Aldrich, H0887), antibiotics (Gibco, 15240096) and EGTA 2 mM] was added and the suspension was mixed gently. Then cells were centrifuged at 800g for 10 minutes at 4oC. Supernatant was removed with a serological pipette and cells were resuspended in HBSS++. Viability was tested using trypan blue (Sigma-Aldrich, T8154) (1:1 dilution) and presence of fluorescence positive cells was evaluated using a normal fluorescence microscope. Just before sorting, cells were passed through a 100 µm BD Filcon (BD Biosciences, 340610) in order to remove cell aggregates. For E16.5 lungs, after digestion, centrifuge and cell resuspension in HBSS++, cells were passed through a 100 µm BD Filcon (BD Biosciences, 340610) and counted. We resuspented the cells in the proper amount of HBSS++ in order to obtain 20x106 cells/ml cell concentration. 100 µl of cell suspension were stained with 0.5 µl of anti-EpCam-PE antibody (Biolegend, 118205) and 0.5 µl anti-CD45-APC antibody (Biolegend, 103112). Replicates of the above reactions were set in separate tubes; in order to prevent the formation of cell aggregates, which is typical when we centrifuge large amount of epithelial cells. For cell-sorting, we used a BD FACSARIA III cell-sorter, using 100 µm nozzle, applying single-cell sorting purity setting. For E16.5 lungs, EpCampos CD45neg cells were sorted. Cells from all other stages were isolated according to GFP expression. Cell sorting and single-cell library preparation were done according to Smart Seq2 protocol (Picelli et al., 2013; Picelli et al., 2014) with some modifications. Cells were sorted in 96-well plates (Piko PCR Plates 24-well, Thermo Scientific, SPL0240 and Plate Frame for 24-well PikoPCR Plates, Thermo Scientific, SFR0241).  Each well contained Triton-X100 (0.2%) (Sigma-Aldrich, T9284-100ML), ERCC RNA Spike-In Mix (1:400.000) (Life-Technologies, 4456740), Oligo-dT30 VN (1,25uM) AAGCAGTGGTATCAACGCAGAGTAC (30 x T) VN, dNTPs (2.5mM/each) (Thermo Scientific-Fermentas, R0192) and Rnase Inhibitor (1U/ul) (Clontech, 2313A) in 4 µl final volume. After sorting, strips were covered with Axygen PCR-tube caps (VWR, PCR-02-FCP-C), centrifuged and placed on dry ice until storage at -80oC for further use. For cell culture cells were sorted into HBSS++ buffer and kept on ice until they were placed in culture. Smart Seq2 protocol is optimal for cell lines and/or large human cells (Picelli et al., 2014). Mouse primary pulmonary cells are smaller and they contain smaller amount of RNA, as a result high amount of primer concatamers were formed during cDNA library preparation, compromising its efficiency. Our optimized protocol contains 50% less Oligo-dT30 VN and cDNA synthesis was divided into two steps, the first was without TSO LNA and the second contained 1µM TSO LNA and additional 40 U of SuperScript II RT (Thermo-Fisher Scientific, 18064071). Reaction lasted 30 minutes at 42oC. Then enzyme was deactivated at 70oC for 15 minutes. For Pre-Amplification PCR, we used the KAPA HiFi Hotstart ReadyMix (2x) (KAPA Biosystems, KK2602) and the ISPCR-primer AAGCAGTGGTATCAACGCAGAGT. PCR included 21 cycles and the total volume increased to 50ul in order to reduce the concentration of the unused Oligo-dT30 VN and TSO-LNA primers. Tagmentation and indexed library amplification were done with Nextera® XT DNA Library Preparation Kit (Illumina, FC-131-1096) and Nextera® XT Index Kit (96 indexes, 384 samples), (Illumina, FC-131-1002) according to the manufacturer protocol (with 2.5 x volume reduction in all reactions). The quantity of the used library for tagmentation was 50 pg of the 500-9000 bp fraction of the library, as indicated by its Bioanalyzer profile.