GSM6480963: Sample_Glucan_84_pool3C1; Mus musculus; RNA-Seq
Cells of the bronchoalveolar space were harvested by flushing the lungs with 3x 1 ml ice cold 1x PBS containing 10 mM EDTA. After centrifugation with 1350 rpm for 5 min at 4°C, cell pellets were resuspended in antibody mix and stained for 35 min at 4°C. After washing with FACS buffer (1x PBS, 2 mM EDTA, 0.5% BSA (SERVA)) Seq-Well libraries were generated as described by Gierahn et al. (Gierahn et al., 2017). Briefly, 110000 barcoded mRNA-capture beads in Bead Loading Buffer were loaded onto the array. Afterwards, the Bead Loading Buffer was replaced by RPMI 1640 medium (Gibco) with 10% FCS (Sigma-Aldrich) and incubated for 10 min. The medium was discarded, 20000 to 30000 BALF cells were loaded and rocked for 10 min. The loaded arrays were washed 5x with PBS, soaked in RPMI 1640 und sealed by polycarbonate membranes under mild vaccum. The sealed arrays were incubated for 37°C for 30 min in Agilent clamps (Agilent) and then incubated in a guanidinium-based lysis buffer for 20 min. After incubation in hybridization buffer, the mRNA capture beads were washed from arrays and collected. Reverse transcription was performed on the bead pellet using a Maxima Reverse Transcriptase reaction (ThermoFisher) for 30 min at room temperature with end-over-end rotation followed by 90 min incubation at 52°C with end-over-end rotation. The reaction was stopped by washing with TE buffer supplemented with 0.01% Tween-20 or 0.5% SDS. Excess primers were digested by exonuclease ExoI (New England Biolabs). Beads were counted and the reverse transcribed cDNA libraries were amplified in a PCR reaction. After PCR, 20000 – 40000 beads were pooled and cleaned using AMPure XP beads (Beckman Coulter). The library integrity was assessed using a High Sensitivity D5000 assay (Agilent) for Tapestation 4200 (Agilent).