5 protocols
AccessionType
nucleic acid sequencing protocol
Samples were sequenced on an Illumina HiSeq 2000 machine. Single-end 50 bp reads were used.
nucleic acid library construction protocol
Single-cell or technical replicate cDNA libraries were prepared as described previously (Tang et al. 2010 Nat Protoc 5(3): 516-535) with the following modifications: PBS had to be replaced by solution A for plant cells because A. thaliana cells rapidly die in PBS. We confirmed that solution A does not negatively affect the performance of the protocol. Furthermore, we included a third RNase inhibitor from Qiagen (#129916), used 1 ul of the reverse transcription master mix, and performed 24 cycles of the initial PCR. HeLa total RNA and ERCC spike-ins were diluted using RNase free water. 1 ul of a 1:1,000,000 dilution of the ERCC spike-ins and 50 pg of HeLa total RNA were included in the lysis buffer. Final cDNA libraries (200ng - 2000ng depending on cell type) were checked for known marker genes, and after passing quality control, libraries were fragmented with the Covaris S2 system as reported previously (Tang et al. 2010 Nat Protoc 5(3): 516-535) using Covaris mircoTUBEs (#520045) and a volume of 130 ul for shearing (libraries were diluted to that volume using RNase-free water). After fragmentation, the volume was reduced to 85ul using a SpeedVac concentrator and samples were subjected to standard Illumina library preparation using the NEBNext DNA Sample Prep Master Mix Set 1 kit according to manufacturers instructions. Modified Illumina PE adapters including custom-made multiplexing barcodes were ligated (amount of adapters was adjusted according to amount of input material), and Illumina PE primers (PE PCR Primer 1.0 and PE PCR Primer 2.0) were used for the PCR enrichment step (10 cycles) of the NEBNext protocol. The final purification step was performed using AMPure XP beads rather than columns and clusters were generated following the standard Illumina protocol.
sample treatment protocol
QC and Gl2 cells were isolated as previously described (Birnbaum et al. 2005 Nat Methods 2(8): 615-619). In brief, root tips of fluorescent marker lines were cut off using a scalpel and transferred to solution B for protoplasting (0.6 M Mannitol, 10mM KCl, 10 mM MgCl2, 10 mM CaCl2, 1 mg/ml BSA, 0.39 mg/ml MES, pH 5.5, 1.5% Cellulase R10, 0.1% Pectolyase Y-23). Root tips were protoplasted on a platform shaker for 30 min for the Gl2 cells and 60 min for the QC cells. Release of QC cells was facilitated by gently streaking root tips on a 75 um cell strainer every 15 min whereas GL2 cells were released by gently pipetting pieces of tissue up and down using a mouth pipette. Single cells of interest were identified by GFP signal and washed 3 times using individual drops of washing solution A (0.6 M Mannitol, 10 mM KCl, 10 mM MgCl2, 10 mM CaCl2, 1 mg/ml BSA, 0.39 mg/ml MES, pH 5.5). After 3 washes cells were transferred to PCR tubes containing the appropriate lysis buffer within a volume > 0.5 ul of solution A.
growth protocol
The pWOX5::GFP and pGL2::GPF seeds where sterilized using 4 h of standard bleach vapor sterilization (Clough and Bent 1998 Plant J 16(6): 735-743). After sterilization seeds were stratified in the dark at 4C for 72 h and plants were subsequently grown vertically for 4 days in constant light conditions at 22C and 45% humidity. Standard root plates were used for growing plants (i.e., 1 x Murashige and Skoog basal medium, 0.5% sucrose, 2.6 mM 2-(N-Morpholino) ethanesulfonic acid (pH 5.7) and 1% agarose).
nucleic acid library construction protocol
A. thaliana total RNA was diluted using RNase-free water, and 50 pg were used for each cDNA library preparation using the SMARTer Ultra Low RNA Kit for Illumina Sequencing from Clontech according to manufacturers instructions. 24 cycles of PCR were used for library amplification, and after passing quality control, finalized cDNA libraries (6 ng - 20 ng) were fragmented with the Covaris S2 system as reported previously (Tang et al. 2010 2010 Nat Protoc 5(3): 516-535) using Covaris mircoTUBEs (#520045) and a volume of 130 ul for shearing (libraries were diluted to that volume using RNase-free water). After fragmentation, the volume was reduced to 44 ul using a SpeedVac concentrator and samples were subjected to standard Illumina library preparation using the NEBNext ChIP-Seq Sample Prep Master Mix Set 1 kit according to manufacturers instructions. Illumina PE adapters including custom-made multiplexing barcodes were ligated (amount of adapters was adjusted according to amount of input material), and Illumina PE primers (PE PCR Primer 1.0 and PE PCR Primer 2.0) were used for the PCR enrichment step (15 cycles) of the NEBNext protocol. The final purification step was performed using AMPure XP beads rather than columns and clusters were generated following the standard Illumina protocol.