title: dilution_10pg
organism: Arabidopsis thaliana
description: Protocols: 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2013-03-21T17:02:36Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:17:27Z
attributes.tag: External Id
attributes.value: SAMEA1713718
attributes.tag: INSDC center name
attributes.value: EMBL-EBI
attributes.tag: INSDC first public
attributes.value: 2013-03-21T17:02:36Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:17:27Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1499:dilution_10pg
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: thale cress
attributes.tag: individual
attributes.value: dilution_10pg
attributes.tag: organism part
attributes.value: leaf
attributes.tag: sample name
attributes.value: E-MTAB-1499:dilution_10pg
attributes.tag: scientific_name
attributes.value: Arabidopsis thaliana