title: GSM2695776: CD166posCD24neg_Negative_S45; Mus musculus; RNA-Seq
library_construction_protocol: A 1.8X volume of AMPure beads (Beckman Coulter, Brea, CA) was added to the thawed cell lysates (~10,000 cells sorted into 500ul Lysis Buffer RLT (Qiagen, Valencia, CA)). After a 30 minute incubation at room temperature, the samples were washed 2X with 70% ethanol and eluted in 22 µl water.  The samples were then digested with 0.6 mAU Proteinase K (Qiagen, Valencia, CA) in the presence of 1X NEB Buffer 1 (New England Biolabs, Ipswitch, MA) at 50C for 20 minutes, followed by a heat inactivation step at 65C for 10 minutes. A DNase digestion was performed using the RNase-Free DNase Set (Qiagen, Valencia, CA) at 37C for 30 minutes. The samples were cleaned with a 1.8X volume of AMPure XP beads (Beckman Coulter, Brea, CA). 1 ng of purified total RNA, as determined by Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA), was processed with the mRNA direct micro kit (Life Technologies, Foster City, CA) to select for poly A RNA. Each entire sample was input into the Ambion WT Expression Kit (Life Technologies, Foster City, CA) to perform double stranded cDNA synthesis followed by in vitro transcription to generate amplified cRNA. The cRNA was purified following the manufacturer’s instructions and the concentration was determined by a NanoDrop instrument (Thermo Fisher Scientific, Waltham, MA).  1 µg of cRNA was fragmented in 1X Fragmentation Buffer (mRNA-seq Sample Prep Kit, Illumina, Hayward, CA) at 94C for 5 minutes, then placed on ice and the reaction was stopped by the addition of 20 mM EDTA. The fragments were precipitated with 70mM Sodium actetate (Life Technologies, Foster City, CA), 40 µg glycogen (Life Technologies, Foster City, CA) and 70% ethanol at -80C for 1 hour followed by centrifugation and washing with 70% ethanol.  3 µg of random hexamer (Life Technologies, Foster City, CA) was added to the fragmented, purified cRNA and incubated at 70C for 10 minutes to anneal the primer. The first strand reaction was performed with 200 units of SuperScript II (Life Technologies, Foster City, CA) with 0.625 mM dNTP’s (NEB, Ipswitch, MA) and 8U SUPERase RNase Inhibitor (Life Technologies, Foster City, CA) at 25C for 10 minutes, then 42C for 50 minutes, then 75C for 15 minutes and cooled to 4C. In second strand synthesis, 1X second strand buffer (Illumina, Hayward, CA) and 0.3mM dNTP’s  (Illumina, Hayward, CA) were added and the samples were incubated at 4C for 5 minutes before adding 50 units of DNA Polymerase (NEB, Ipswitch, MA) and 5 units of Rnase H (NEB, Ipswitch, MA). The samples were mixed well and incubated at 16C for 2.5h, followed by purification with the MinElute Kit (Qiagen, Valencia, CA). To perform library prep, the samples were end repaired using a Quick Blunting Kit (NEB, Ipswitch, MA) and incubated at 20C for 1 hour, then 75C for 30 minutes to inactivate the enzyme. A’ bases were added to the 3’ ends of each fragment with 2 mM dATP and 5 units of Klenow fragment 3’-5’ exo- DNA Polymerase (NEB, Ipswitch, MA) at 37C for 45 minutes, followed by 75C for 30 minutes to inactivate the enzyme. Using a quick ligase kit (NEB, Ipswitch, MA), 0.5uM of adaptors were ligated to the cDNA fragments at 12C for 75 minutes, then 80C for 20 minutes and cooled to 4C. These adaptors contain barcodes to facilitate sample multiplexing during sequencing. The adaptor sequence is preceded by four random nucleotides to add diversity to the pooled library. The samples were pooled by combining 5 µl of each library. After AMPure XP cleanup, one half of the pooled library was run on the Pippin Size Selection Instrument (Sage Sciences, Beverly, MA) to select for 200bp fragments. Library amplification was performed on one half of the Pippin eluate in 1X Phusion GC buffer with 0.2mM dNTP’s, 0.1uM forward primer (IDT, Coralville, IA), 0.1 µM reverse primer, 1 unit of Phusion Hot Start II Polymerase (Thermo Fisher Scientific, Waltham, MA). The reaction was run with the following program: 98C for 30s, then 15 cycles of 98C for 10s, 65C for 30s, 72 for 30s, then 72C for 4 minutes and cooled to 4C. The amplified library was cleaned using a 1X volume of AMPure XP beads and QC was run with the Agilent Bioanalyzer DNA 1000 kit, followed by concentration determination by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). In order to qualify for sequencing, the library must have a minimum concentration of 4nM and have no trace of low MW (<150bp) primer/dimer product.
attributes.tag: GEO Accession
attributes.value: GSM2695776