Illumina HiSeq 2000 sequencing; GSM1820357: Run283_L4_A03_single cell; Mus musculus; RNA-Seq

Run283_L4_A03_single cell

We obtained full transcriptome data from single cortical neurons after whole-cell patch-clamp recording (termed “Patch-seq”). By applying “Patch-seq” to cortical neurons, we reveal a close link between biophysical membrane properties and genes coding for neurotransmitter receptors and channels, including well-established and hitherto undescribed subtypes. Overall design: RNA sequencing was performed on a total of 83 individual cells

Single-cell RNA sequencing of aspirates from cortical neurons after patch clamp recording

Submitted by Gene Expression Omnibus on 22-DEC-2015

At the end of each patch-clamp protocol, the micropipette was clamped to a holding potential. The entire soma of each recorded neuron was aspirated into the micropipette slowly by applying negative pressure, and ejected into a drop of lysis buffer. 20 microL lysis buffer (0.15% Triton X-100, 1 U/microL TaKaRa RNase inhibitor, 4 microM 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 microM template-switching oligo C1-P1-RNA-TSO, 1.5 U/microL TaKaRa RNase inhibitor and 18 U/microL Life Technologies Superscript II reverse transcriptase) and PCR mix (1.1x Clontech Advantage2 PCR buffer, 440 microM 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 microL C1 Harvesting Reagent and quantified on an Agilent BioAnalyzer. The typical yield was 1 ng/microL. 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 microM barcoded adapter C1-TN5-x, 40% glycerol, 6.25 microM Tn5 transposase, where x denotes a well-specific barcode) were prepared, each with a different barcode sequence.  6 microL harvested cDNA was mixed with 5 microL tagmentation buffer (50 mM TAPS-NaOH pH 8.5, 25 mM MgCl2 and 50% DMF), 11.5 microL nuclease-free water and 2.5 microL 10x transposome stock. The mix was incubated for 5 minutes at 55 degeees C then cooled on ice. 100 microL 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 microL 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 microL TNT (20 mM Tris pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 microL Qiagen Qiaquick PB, then twice in 100 microL TNT. The beads were then resuspended in 100 microL restriction mix (1x NEB NEBuffer 4, 0.4 U/microL PvuI-HF enzyme), designed to cleave 3' fragments carrying the PvuI recognition site. The mix was incubated for one hour at 37 degrees C, then washed three times in TNT. Finally, the single-stranded library was eluted by resuspending the beads in 100 microL 100 mM NaOH, incubating for 5 minutes, removing the beads, then adding 100 microL 100 mM HCl and 50 microL 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 were rom these were run on  with 187ds_).

Single-cell RNA sequencing of aspirates from cortical neurons after patch clamp recording

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