title: Illumina HiSeq 2500 sequencing; Mouse embryonic RNA-seq
design: Mouse embryonic RNA-seq
library_construction_protocol: Timed matings were set up between CD1 mice. Embryos were staged according to morphologic criteria (PMID:8269852). Single cell suspensions from individual embryos were prepared by incubating embryos with TrypLE Express dissociation reagent (Life Technologies) at 37C for 10 minutes and quenching with heat-inactivated serum. The sample was then sorted using a BD Influx in single cell mode. Single cells were sorted into individual wells of a 96 well PCR plate containing lysis buffer from the scRNA-Seq protocol (see RNA extraction protocol).  At E6.5, caudal halves of embryos were dissected and dissociated to capture only epiblast cells, and cells were sorted only on the basis of viability.  From E7.0 onwards, whole embryos were dissociated.  At E7.0-7.75, cells were stained for Flk1 and Flk1+ cells were sorted.  At E7.5 and E7.75, cells were also stained for CD41 and Flk1+CD41+ and Flk1-CD41+ populations were also sorted. scRNA-Seq protocol was adapted from SmartSeq2 (PMID: 24385147).  All samples should be processed in a pre-amp setting, inside a UV-laminar hood. Single cells were sorted in 2.3 l of lysis buffer (0.2% Triton x-100 and 1 U RNase Inhibitor). 2 l of annealing mix was added per well (ERCCs, 5 M oligo-dT, 5 mM dNTPs, dH20). Samples were then incubated at 72oC for 3 minutes and immediately placed on ice. 5.7 l of reverse transcription mix was added per well (17.5 U Superscript II RT, 0.9 U RNase Inhibitor, 1.75 x First stand buffer, 8.77 mM DTT, 1.75 M Betaine, 0.01 M MgCl2, 1.75 M TSO and dH2O). Reverse transcription was performed (1) 42oC for 90 mins, 2) 50oC for 2 mins, 42oC for 2 mins (10 cycles), 3) 70oC for 15 mins, 4) 4oC hold). Add 15 l PCR mix (1.6 x KAPA HiFi Hotstart Ready Mix, 0.17 M IS PCR primer and dH2O), PCR was then performed (1) 98oC for 3 mins, 2) 98oC for 20 secs, 67oC for 15 secs, 72oC for 6 mins (21 cycles), 3) 72oC for 5 mins and 4) 4oC hold). All products are now considered to be Post-amplification and should be handled in a Post-PCR setting. Samples were then cleaned up using Ampure XP beads at a 1:1 ratio. The quality of the library was then assessed on the Agilent Bioanalyser. Sequencing libraries were then prepared using the Illumina Nextera kit according to the Fluidigm protocol. The wells were then pooled and cleaned up using Ampure XP beads at a 1:0.9 ratio. The amplified libraries were then checked for quality and size using the Agilent Bioanalyser. Illumina Nextera XT DNA preparation kit  Sequencing libraries were prepared using the Illumina Nextera kit according to the Fluidigm protocol. The wells were then pooled and cleaned up using Ampure XP beads at a 1:0.9 ratio. The amplified libraries were then checked for quality and size using the Agilent Bioanalyser.
attributes.tag: Experimental Factor: embryonic day 7.5
attributes.value: developmental stage
attributes.tag: Experimental Factor: CD41-positive, Flk1-negative
attributes.value: cell type
attributes.tag: Experimental Factor: SLX.8345.N703_N504
attributes.value: single cell ID