GSM5332960: ELSK_3 [R3]; Mus musculus; RNA-Seq
Haematopoietic Stem Cells (HSCs) were extracted from primary mouse bone marrow, derived from C57BL/6 mice(12-16 weks) for scRNA-seq and Fgd5(ZsGreen•ZsGreen/+) for bulk RNA-seq. To isolate fetal liver HSCs, fetal livers were harvested from E14.5 embryos. All extracted tissues were lineage depleted prior to FACS sorting. HSCs were defined as EPCR-high, CD45+, Sca-1+, CD48-low/neg, CD150+. Expansion cultures were FACS sorted to separate ELSK and nonELSK cell fractions. HSCs in hibernation conditions and freshly isolated fetal liver were FACS sorted to be deposited in 96 well plates (one cell per well), containing lysis buffer and subsequently processed using the previously described Smart-Seq2 protocol (Picelli et al., 2014). Smart-Seq2 protocol performed as previously described (Picelli et al., 2014). Libraries were prepared using Illumina Nextera libraries, single end reads and 50bp read lengths. The pooled libraries were sequenced on an Illumina HiSeq 4000. Bulk RNA-seq libraries were prepared using the SMARTer Stranded Total RNA-seq Kit v2 – Pico Input mammalian (Takara Bio, CA, USA), paired end and 50bp read length. The libraries were sequenced on an Illumina Novaseq.