title: GSM5555247: Endometrial MPRA rep 3 [endo_3]; Homo sapiens; OTHER
library_construction_protocol: Total RNA and DNA representing roughly 12 million cells were isolated using Qiagen's AllPrep DNA/RNA Mini Kit. mRNA was then isolated from the total RNA using Dynabeads mRNA DIRECT purification kit (Thermo Fisher) Reactions containing 500ng mRNA and 100nM of a gene-specific primer containing a sample index and UMI (GGW610-501 – GGW610-508, Table S5) were reverse transcribed using Superscript IV (Thermo Fisher), along with a no reverse transcriptase control. Reactions were incubated with 1ul thermolabile exonuclease I (NEB) for 10 min at 37C, followed by heat inactivation for 5min at 85C. The cDNA was cleaned with Ampure XP beads (Beckman Coulter) at a ratio of 1:1.1 sample to beads and eluted in the initial volume. The cDNA was amplified in 50ul reactions containing 5ul cDNA, LK612 at 80nM, one of LK134-701-LK134-712 at 80nM (Table S5), SYBR green (Thermo Fisher), and PrimeStar Max DNA Polymerase (Takara Bio). The reactions were amplified until they were just beginning to rise, then they were pooled and concentrated. The libraries were then gel purified and quantified using the KAPA library quantification kit (Roche). The 42 samples were put into 3 pools which were sequenced using an Illumina NextSeq Sequencer.