title: GSM9436981: Male-Vehicle_TYC_STARR8_RNA_replicate1; Mus musculus; OTHER
library_construction_protocol: The extraction methods, adapted from the published STARR-seq protocol (Neumayr, Pagani et al. 2019), were used in a nested PCR amplification protocol to characterize the transcriptional activity of each DHS sequence (putative regulatory region) to be assayed by HDI-STARR-seq: 1) a library prepared from the same batch of purified plasmid DNA injected into each mouse by HDI, which was used directly as a PCR template in a nested PCR reaction for library construction (primers ON8381, ON8382; see STARR_seq_primer.txt); 2) a library prepared from DNA extracted from flash frozen liver tissue collected 10 days after in vivo transfection by HDI (primers ON8381, ON8382); and 3) a library prepared from the transcribed RNA (reporter sequences) extracted from each HDI-transfected liver (primers ON8379, ON8380; see STARR_seq_primer.txt) . STARR-seq reporter plasmid DNA remaining in the liver 10 days after HDI was extracted using a commercial QIAprep spin miniprep kit (QIAgen, cat. # 27106). The liver tissue (200 mg frozen liver) was initially homogenized in 750 ul of QIAgen Buffer P1 using a rubber-tipped micropestle and passed through a 20G needle three times to homogenize large tissue pieces. The extracted plasmid DNA was bound to a QIAprep spin column, eluted in EB buffer and used as a PCR template in a subsequent nested PCR reaction using primers ON8381 and ON8382 (see STARR_seq_primer.txt). To extract transcribed reporter RNA, 120 ug of total liver RNA was isolated from 60-100 mg liver tissue from each mouse by TRIzol guanidinium thiocyanate-phenol-chloroform extraction then purified by polyA selection using oligo(dT) beads (New England Biolabs, cat. #E7490L). The transcribed RNA was digested with Turbo DNase I (Ambion, cat. # AM2238) at 37°C for 30 min, reverse transcribed using primer ON8731 (see STARR_seq_primer.txt) for STARR-seq RNA first strand synthesis and Superscript III first-strand synthesis supermix (Invitrogen, cat. # 18080-400), and treated with 10 mg RNase A per ml (Fermentas, cat. # EN0531) for 1 h at 37 degrees Celsius. The cDNA obtained was purified using 1.8x SPRI beads then used as a PCR template in the nested PCR reaction. The nested PCR requires one set of primers specificially targeting DNA or RNA form of STARR-seq reporter followed by generalized illumina dual index primers. A primer set, ON8381 and ON8382 (see STARR_seq_primer.txt), was designed to amplify the STARR-seq plasmid DNA libraries by specifically targeting the unspliced intron incorporated into original plasmid and present in the plasmid and DNA libraries but spliced out of the reporter RNA sequences. Another primer set, ON8379 and ON8380 primer (see STARR_seq_primer.txt), which straddles the splice junction, was designed to specifically amplify only the transcribed and spliced RNA molecules. The nested PCR was completed by using  NEBNext® Multiplex Oligos for Illumina (#E7600S) for all three library types (plasmid, DNA and RNA) to further amplify the material and attach Illumina i5 and i7 barcoded sequences to allow for multiplex sequencing on an Illumina sequencer (Novogene, Sacramento, CA).