GSM5233836: FAIRE_STARR_RA_rep3; Mus musculus; FAIRE-seq
As input material for the reporter screen, DNA fragments representing accessible chromatin from E14 mESCs were used. Prior to DNA isolation, the cells were treated with retinoic acid (RA, Sigma, #R2625) for 4h. Accessible DNA was isolated by formaldehyde-assisted isolation of regulatory elements (FAIRE, Giresi et al., 2007) and subsequently cloned into the STARR-seq screening vector (Addgene #71509) following the protocol described in Arnold et al., 2013. E14 cells were transfected with this plasmid library using a NucleofectorTM 2b device applying the Mouse ES Cell Nucleofector Kit (Lonza, VAPH-1001). For each of the three biological replicates, four individual transfections, each with 5 µg plasmid library and 5x10e6 cells, were performed. The medium was changed 12 h after transfection and to half of the cells either LIF or 1 µM RA was added. After an additional 4 h of incubation, RNA was isolated from the combined transfections (of the same treatment) using the RNeasy Midi kit (Qiagen). The polyA RNA fraction was enriched using DnyabeadsTM Oligo(dT)25 (Invitrogen), residual DNA was digested using Turbo DNase (Invitrogen), and finally RNA was cleaned-up with Agencourt® RNAClean® XP beads (Beckman Coulter). cDNA was synthesized using SuperScriptTM III Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol, applying a reporter transcript-specific primer. This primer also includes the sequence of the Illumina PCR Primer 2.0 as overhang as well as eight randomly generated nucleotides that serves as unique-molecular identifier (UMI) to each cDNA molecule (CAAGCAGAAGACGGCATACGAGAT[N]8GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT).  cDNA was further amplified as described in Arnold et al., 2013, using adjusted reporter-specific primers based on Illumina's TruSeq dual index system (universal: CAAGCAGAAGACGGCATACGA, sample specific: AATGATACGGCGACCACCGAGATCTACAC[barcode, n=6]ACACTCTTTCCCTACACGACGCTC).  As input control for FAIRE-STARR-seq, the input plasmid library was sequenced as well. To this end, the plasmid library was used for a pseudo “cDNA synthesis”, using the random-UMI primer and the KAPA HiFi HotStart ReadyMix (Roche) for 4 cycles with a prolonged synthesis step (70 sec) to individually label input fragments. Input and FAIRE-STARR-seq libraries were amplified with Illumina's TruSeq dual index based universal and barcoded primers applying the KAPA HiFi HotStart ReadyMix (Roche) for 12 PCR cycles.