title: GSM2171824: M4-1; Homo sapiens; OTHER
library_construction_protocol: Kapa High Fidelity Polymerase was used for all barcode amplification steps. Gradient PCRs were performed to optimize annealing temperatures for amplification from gDNA. For experiments performed without UMIs, up to 250 ng of gDNA was loaded into a single 50 µl PCR reaction and amplified using primers immediately flanking the barcode (see table S4 for oligo sequences). If there was less than 250 ng from a sample, all of it was used in a single reaction. For experiments performed with UMIs, a primer with a sequencing adapter and 10 nt of fully degenerate sequence 5' to the barcode-flanking sequence was used for a single prolonged extension step, in which the temperature was ramped between annealing and extending for five cycles (without a denaturing step to prevent re-sampling of gDNA barcodes). All cell culture experiments and v6 zebrafish embryos received a single extension to incorporate UMIs, whereas v7 embryo time-course experiments and all ADR1 tissues (also v7) received 2 UMI incorporation cycles due to having low gDNA consequent to fewer cells being present in early embryo and sorted heart samples. To minimize repetitive amplification of the same barcode, no reverse primer was included in UMI-tagging reactions. DNA was then purified using AMPure beads (Agencourt), and loaded into a PCR primed from the sequencing adapter flanking each UMI and a site immediately 3' of the barcode. For all experiments, two ensuing qPCRs were performed prior to sequencing to incorporate sequencing adapters, sample indexes, and flow cell adapters. AMPure beads were used to purify PCR products after each reaction.
attributes.tag: GEO Accession
attributes.value: GSM2171824