Hi!
I am new to the field and I try to establish single cell transcriptome sequencing with the Fluidigm C1 machine. We already decided that we use the SmartSeq2 protocol from Clontech (SmartSeq v4). Now our Institute got rid of several Illumina HiSeq 2000 machines that are recommended to use with the Nextera XT librabry prep that is recommended from Clontech. We do have several HiSeq 4000 machines and after talking with the facility they mentioned I should fractionate the cDNA on a Kovaris (with ultrasound) and then use the NEBNext ChIP Seq Mastermix for Illumina sequencing. Do you think this is the best protocol or is there another available? Of course I checked online but that did not yield much... I checked several papers for single cell sequencing and practically all of them use the Illumina 2000 sequencer. Is there a specific non-obvious reason for that? Is it only because of the Nextera XT Kit?
Unrelated to the SmartSeq2 protocol, can one use UMIs with the Kovaris ultrasound shredding of the cDNA? Will there not be many UMIs lost since they are always at the end of the molecule, so they are on smaller cDNA fragments. Are smaller fragments then more likely to be lost during purification processes?
Thank you very much in advance!
Stephan
I am new to the field and I try to establish single cell transcriptome sequencing with the Fluidigm C1 machine. We already decided that we use the SmartSeq2 protocol from Clontech (SmartSeq v4). Now our Institute got rid of several Illumina HiSeq 2000 machines that are recommended to use with the Nextera XT librabry prep that is recommended from Clontech. We do have several HiSeq 4000 machines and after talking with the facility they mentioned I should fractionate the cDNA on a Kovaris (with ultrasound) and then use the NEBNext ChIP Seq Mastermix for Illumina sequencing. Do you think this is the best protocol or is there another available? Of course I checked online but that did not yield much... I checked several papers for single cell sequencing and practically all of them use the Illumina 2000 sequencer. Is there a specific non-obvious reason for that? Is it only because of the Nextera XT Kit?
Unrelated to the SmartSeq2 protocol, can one use UMIs with the Kovaris ultrasound shredding of the cDNA? Will there not be many UMIs lost since they are always at the end of the molecule, so they are on smaller cDNA fragments. Are smaller fragments then more likely to be lost during purification processes?
Thank you very much in advance!
Stephan
Comment