Reaction cleanup kit

Total RNA was purified from transduced cells (Qiagen #75144) and concentrated 7.5X using an RNA Cleanup Kit (NEB #T2040L). RNA was reverse transcribed according to manufacturer guidelines with STARR-Seq transcript–specific primers (Supplemental Table 1) with SuperScript IV (Invitrogen #18090010), using twice the recommended RNA input. cDNA was purified using a Reaction Cleanup Kit (Qiagen #28206) and amplified using custom STARR-Seq transcript–specific primers (Supplemental Table 1) as previously described [15 (link)]. Diluted Lenti-STARR-Seq plasmid ‘input’ control was amplified using an input concentration that yielded the same amplification cycle number as the STARR library for hCD4+ Donor I. PCR amplified library was purified using PCR cleanup columns (Qiagen #28206) and quantified for PE-150 bp sequencing (Novogene).

The transcription start site (TSS) of the resR/mcdR mRNA was determined by the 5’-RACE (rapid amplification of cDNA ends) technique, as described previously^{22 ,45 (link)}. Briefly, the DNase-treated RNA was reverse transcribed using resR/mcdR_RT-R primer (Supplementary Data 4), followed by 30 minutes of incubation at 37 °C with RNase H and RNase A. The cDNA was subsequently purified using reaction clean up kit (Qiagen), and subjected to dC tailing with the help of dCTP and terminal transferase (NEB). The dC-tailed cDNA was PCR amplified using Abridged Anchor (Thermo Fisher) and resR/mcdR_RT-R primer pair (Supplementary Data 4). The nested PCR was performed using Abridged Anchor and resR/mcdR_CrUP primer pair (Supplementary Data 4). The PCR products were subsequently sequenced using resR/mcdR_CrUP.

3Flag-Cse4-containing nucleosomes were isolated by ChIP of 3Flag-Cse4 using monoclonal anti-Flag M2 antibodies (Sigma-Aldrich Catalog # F3165). ChIPs were performed with Micrococcal nuclease (MNase, Worthington Biochemical Corporation Catalog # LS004798)-treated chromatin as described [55 (link)] with the following addition. Before nuclei isolation, proteins were crosslinked to DNA with 1% formaldehyde for 15 minutes. Crosslinks were then reversed before DNA extraction by the addition of 1% SDS and an overnight incubation at 65°C [87 (link)]. DNA was extracted using phenol:chloroform extraction and ethanol precipitation, and was treated with RNAse and purified using a Qiagen Reaction Clean-up kit before library construction. Paired-end sequencing libraries of both input DNA from MNase-digested chromatin and 3Flag-Cse4 ChIP DNA were prepared using a modified Solexa library preparation protocol that captures DNA particles down to ~25 bp [55 (link)]. Cluster generation, followed by 25 cycles of paired-end sequencing on an Illumina HiSeq 2000, was performed by the Fred Hutchinson Cancer Research Center Genomics Shared Resource facility, resulting in 24 bp paired end reads. Base calling was performed using Illumina's Real Time Analysis software v1.13.48.0. Raw FASTQ sequence files were deposited in the NCBI GEO Series GSE69696.