title: GSM3362253: 1772100100_G07; Mus musculus; RNA-Seq
library_construction_protocol: After the recording, weak negative pressure was applied in order to aspirate the recorded cells into the glass capillary patch electrode. Using positive pressure the pipette content was quickly ejected into a 1 μL drop of RNase-free lysis buffer. placed on the side of a 0.2 mL RNase free tight-lock tube (TubeOne). The sample was rapidly spun down (5-10 s) and stored at −80 before reverse transcription (RT). 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 wells. The plate was put in 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_).
attributes.tag: GEO Accession
attributes.value: GSM3362253