Journal: bioRxiv

Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

doi: 10.1101/099093

Figure Lengend Snippet: Emulsion RT-PCR can be used to join barcodes into barcode pairs. (a) Schematic of emulsion, in-drop RT and overlap PCR followed by breaking of the emulsion and purification of barcode pairs. (b) Dilution of input RNA decreases the false positive rate of emulsion PCR. Two separate barcode pairs (A-A and B-B) are subjected to uniform emulsions simultaneously to probe the concentrations at which overlap PCR becomes promiscuous. Fraction = 0.5 is equivalent to an aqueous (non-emulsified) PCR reaction. Here RNA molecules containing both a pre- and post- segment were employed. (c) “Connectivity matrix” obtained from emulsion RT-PCR followed by Illumina sequencing of synPRE-P—synPOST-P complexes from HEK cell culture, demonstrating that in principle SYNseq can be used to reconstruct synaptic circuits.

Article Snippet: Stability through thermocycling was further increased by adding Tetronic 1307 (BASF) at 1 % weight to the RT-PCR mixture [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Purification, Sequencing, Cell Culture

Journal: bioRxiv

Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

doi: 10.1101/099093

Figure Lengend Snippet: Schematic of overlap RT-PCR. (a) Pre-RNA and Post-RNA molecules containing the barcode sequences, as well as Solexa I and Solexa II sequences for high-throughput sequencing. (b) The RNA molecules are reverse transcribed to cDNA. (c) cDNA is subjected to second strand synthesis using a primer that adds a region of homology between the preRNA and postRNA. (d) Barcodes are amplified individually via an external primer and a limiting concentration of internal primers. (e) Internal primers become limiting and the preRNA and postRNA amplicons perform overlapping PCR. (f) The fused barcode pairs are further amplified using the external primers, before being subjected to (g) nested PCR amplification using the sequencing primers.

Article Snippet: Stability through thermocycling was further increased by adding Tetronic 1307 (BASF) at 1 % weight to the RT-PCR mixture [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Next-Generation Sequencing, Amplification, Concentration Assay, Nested PCR, Sequencing

Journal: bioRxiv

Article Title: Using high-throughput barcode sequencing to efficiently map connectomes

doi: 10.1101/099093

Figure Lengend Snippet: Emulsion RT-PCR from HEK cells. (a) Schematic of the RNA molecules used to benchmark emulsion RT-PCR performance (e.g. in ). Here a single RNA molecule contains both a pre- and post-segment, but in opposite orientations from what is necessary for a sequence-able barcode pair. All the steps described in are therefore necessary to make this RNA into a barcode pair, but no time consuming immunoprecipitations are necessary. (b) Immunoprecipitated complexes from HEK cells co-transfected with synPRE-P and synPOST-P and crosslinked with the BG-PEG-(S-S)-Biotin-PEG-BC crosslinker can be efficiently released from the beads via the application of DTT. (c) qPCR quantification of overlap between the two zip coded experiments in the HEK cell emulsion RT-PCR. We used a dilution of 1:1000 of the IP input for the droplets to create the “connectivity” matrix displayed in . Further dilution of the IP sample would presumably have resulted in a lower crossover rate, as no asymptotic crossover rate has been reached yet.

Article Snippet: Stability through thermocycling was further increased by adding Tetronic 1307 (BASF) at 1 % weight to the RT-PCR mixture [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Immunoprecipitation, Transfection