title: RNA-Seq Library Generation from Rare Human Cells Isolated by FACS.
abstract: High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted hESCs (n = 3). We predict that multiplexed libraries can also be generated by replacing the single-plex 3' adapter used here with a multiplexing compatible 3' adapter and indexed PCR primers.
authors.affiliation: Molecular Cell and Developmental Biology, University of California, Los Angeles, USA.
authors.forename: Sofia
authors.initials: S
authors.lastname: Gkountela
authors.affiliation: Molecular Cell and Developmental Biology; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, USA.
authors.forename: Amander T
authors.initials: AT
authors.lastname: Clark