GSM1541001: single secretagogin+ cell 4; Mus musculus; RNA-Seq
The PVN region of juvenile mice was isolated from 300-μm-thick coronal slices under microscopy guidance and dissociated using the Papain Dissociation System (Worthington). Isolated single cells were concentrated by centrifugation to a density of 300–500 cells/μL and loaded according to the manufacturer’s protocol on a C1 Single-Cell AutoPrep IFC microfludic chip , and the chip was then processed on a Fluidigm C1 instrument using the “mRNA Seq: Cell Load (1772x/1773x)” script. This captured one cell in each of up to 96 capture chambers, and took approximately 30 minutes. 20 μL lysis buffer (0.15% Triton X-100, 1 U/μL TaKaRa RNase inhibitor, 4 μM reverse transcription primer C1-P1-T31, 5% C1 Loading Reagent and 1:50,000 Life Technologies ERCC  Spike-In Mix 1), reverse transcription mix (1x SuperScript II First-Strand Buffer supplemented with 3 mM MgCl2, 1.5 mM dNTP, 4 mM DTT, 3.3% C1 Loading Reagent, 1.8 μM template-switching oligo C1-P1-RNA-TSO, 1.5 U/μL TaKaRa RNase inhibitor and 18 U/μL Life Technologies Superscript II reverse transcriptase) and PCR mix (1.1x Clontech Advantage2 PCR buffer, 440 μM dNTP, 530 nM PCR primer C1-P1-PCR-2, 5% C1 Loading Reagent and 2x Advantage2 Polymerase Mix) were added to the designated wells according to the manufacturer’s instructions, but using the indicated mixes in place of the corresponding commmercial reagents. The plate was then returned to the Fluidigm C1 and the “mRNA Seq: RT + Amp (1772x/1773x)” script was executed, which took about 8.5 hours and included lysis, reverse transcription and 21 cycles of PCR. When the run finished, the amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent and quantified on an Agilent BioAnalyzer. The typical yield was 1 ng/μL. Amplified cDNA was simultaneously fragmented and barcoded by ‘tagmentation’, i.e. using Tn5 DNA transposase to transfer adapters to the target DNA. 96 different 10x transposome stocks (6.25 μM barcoded adapter C1-TN5-x, 40% glycerol, 6.25 μM Tn5 transposase, where x denotes a well-specific barcode) were prepared, each with a different barcode sequence.  6 μL harvested cDNA was mixed with 5 μL tagmentation buffer (50 mM TAPS-NaOH pH 8.5, 25 mM MgCl2 and 50% DMF), 11.5 μL nuclease-free water and 2.5 μL 10x transposome stock. The mix was incubated for 5 minutes at 55°C then cooled on ice. 100 μL Dynabeads MyOne Streptavidin C1 beads were washed in 2xBWT (10 mM Tris HCl pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) then resuspended in 2 mL 2xBWT. 20 μL beads was added to each well and incubated at room temperature for 5 minutes. All fractions were pooled, the beads were immobilized and the supernatant removed (thus removing all internal fragments, and retaining only the 5'- and 3'-most fragments). The beads were then resuspended in 100 μL TNT (20 mM Tris pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, then twice in 100 μL TNT. The beads were then resuspended in 100 μL restriction mix (1x NEB NEBuffer 4, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3' fragments carrying the PvuI recognition site. The mix was incubated for one hour at 37°C, then washed three times in TNT. Finally, the single-stranded library was eluted by resuspending the beads in 100 μL 100 mM NaOH, incubating for 5 minutes, removing the beads, then adding 100 μL 100 mM HCl and 50 μL Neutralization buffer (200 mM Tris pH 7.5, 0.05% Tween-20). At this point, the typical yield was 1-5 nM single-stranded library. Aliquots from these were run on the Hiseq2000 (rawdata file names prefixed with 187ss_ and 187ss-2_). Another aliquot was further amplified by 9 cycles of PCR to produce a double-stranded library as described in Islam, S. et al. Nat. Protoc. 7, 813-828 (2012). These were also run on the Hiseq2000 (rawdata file names prefixed with 187ds_).