GSM3406532: 1772096100_C02; Mus musculus; RNA-Seq
The tissue pieces were dissociated using the Papain dissociation system (Worthington) following manufacturers recommendations, adjusting incubation time based on tissue piece size to 25-45 mins. Using glass pipettes of increasingly smaller tip diameter, the cell suspension was then filtered with 20um filter (Partec) and cells kept in N2 media, and cells loaded into C1 chips for cell capture. Fluidigm C1 Autoprep System  Cells medium (10-17 micrometer) microfluidic was used to capture the cells. 14 microlitre of cell suspension (approx. 800 cells/microlitre in N2 culturing medium with DNaseI) was mixed with 7 microlitre C1 Suspension Reagent after filtering. Single-cells were then captured for 30 min at 4°C using the тАЬCell Load (1772x/1773x) script. The protocol for Lysis, RT and PCR was performed as previously described  (Islam et al., Nat Methods. 2014 Feb;11(2):163-6). Amplified cDNA was harvested with 13 microlitre Harvest Reagent and cDNA library quality was measured on an Agilent BioAnalyzer. Cell barcoding and fragmentation was performed in a single step using Tn5 DNA transposase as described previously. 1 microlitre Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were resuspended in 20 microlitre Binding and Blocking buffer (10mM Tris, 250mM NaCl, 5mM EDTA, 0.5% SDS) and added to each well. After 15 min incubation at room temperature, all wells were pooled, the beads washed once with 100 microlitre Washing buffer (10mM Tris-150mM NaCl, 0.02% Tween), once in 100 microlitre Qiagen Qiaquick PB and then twice using 100 microlitre Washing buffer. Restriction was performed to cleave 3тАЩ fragments: the beads were incubated in 100 microlitre restriction mix (1x NEB CutSmart, 0.4 U/microlitre PvuI-HF enzyme) for 1h at 37°C. Finally, the beads were washed three times with Washing buffer, then resuspended in 30 µl ddH2O and incubated for 10 min at 70°C to elute the DNA. AMPure beads XP (Beckman Coulter) were used at 1.8x volume and eluted in 30 microlitre to remove short fragments.