10,000 hepatocytes from each sorted population were collected for library preparation. Cells were sorted into Lysis buffer supplied in Dynabeads mRNA Purification Kit (Invitrogen, cat: 61006). RNA was extracted by the kit according to the provided protocol. 2ul of the extracted mRNA from each sample were used for libraries. Library preparation was done with mcSCRBseq protocol66 (link). The cDNA was pre-amplified with 10-15 cycles, depending on cDNA concentration indicated by qPCR quality control. 2ng of the amplified cDNA was converted into sequencing library with the Nextera XT DNA Library kit (Illumina, FC-131-1024), according to supplied protocol. Quality control of the resulting libraries was performed with an Agilent High Sensitivity D1000 ScreenTape System (Agilent, 5067- 5584). Libraries were loaded with a concentration of 2.2pM on 75 cycle high output flow cells (Illumina, FC-404-2005) and sequenced on a NextSeq 500 (Illumina) with the following cycle distribution: 8bp index1, 16 bp read1, 66 bp read2 (no index2 needed). Total 40 libraries, eight sorted populations for five different mice were sequenced.
Protocol detail
Single-cell RNA-seq of Sorted Hepatocytes
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Dynabeads mRNA Purification Kit
Nextera XT DNA Library kit
Agilent High Sensitivity D1000 ScreenTape System
NextSeq 500
Buffer
Cdna
Cells
Cycle cells
Dna library
Fc 131
Hepatocytes
Library
Mice
Mrna
Populations
Sensitivity
Corresponding Organization :
Other organizations : Weizmann Institute of Science
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Variable analysis
independent variables
- Sorted cell populations
- Gene expression profiles of the sorted cell populations
- Number of cells collected for library preparation (10,000 hepatocytes from each sorted population)
- Lysis buffer used for RNA extraction (Dynabeads mRNA Purification Kit)
- Library preparation protocol (mcSCRB-seq)
- Number of cDNA pre-amplification cycles (10-15 cycles)
- Amount of amplified cDNA used for library conversion (2ng)
- Library conversion kit (Nextera XT DNA Library kit)
- Sequencing platform (NextSeq 500)
- Sequencing cycle distribution (8bp index1, 16 bp read1, 66 bp read2)
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