Q2. What was used to sort and index the resulting bam files?
SAMtools 1.5 (H. Li et al., 15 2009) was used to sort and index the resulting bam files, as well as for extracting uniquely mapping reads.
Q3. How long after starting the differentiation was the luciferase 2 activity measured?
24 or 40 h after starting the differentiation, luciferase 2 activity was measured using the Dual-Glo® Luciferase Assay System (Promega).
Q4. What was used to convert the sam files to bam files?
SAMtools 1.5 (H. 13 Li et al., 2009) was used to convert the resulting sam files to bam files, to sort and index the bam files as 14 well as for extracting uniquely mapping reads.
Q5. How many times was the pre-warmed Amplifier Mix added to the cells?
After washing cells three times in 20 wash buffer – again including the 2 min incubation before removing the buffer – Amplifier Mix diluted 21 1:25 in pre-warmed Amplifier Diluent QF was added.
Q6. How did the authors map reads to 20 custom-made bowtie indexes?
To account for the genetic changes in the KI cell lines, the authors mapped reads to 20 custom-made bowtie indexes, in which PE had been removed from its endogenous position, and instead 21 had been reintroduced upstream of the promoter in either sense or antisense orientation.
Q7. What is the effect of distance on enhancer activity?
While enhancer activity is 11 generally believed to be independent of genomic distance and large distances can be overcome by 12 enhancer-promoter loops (Furlong & Levine, 2018), recent studies suggest that enhancer-promoter 13 distance can indeed have an effect on expression levels (Carleton et al., 2017; Scholes et al., 2019).
Q8. How many enhancers could act at the same promoter?
Previous studies in Drosophila have suggested that 21 multiple weak enhancers could act simultaneously at a promoter to achieve higher or super-additive 22 transcription initiation rates compared to individual enhancers (Bothma et al., 2015; Carleton et al., 23 2017).
Q9. How many reads were sequenced to a depth of 30-60 million reads?
Sequencing was performed at the VBCF NGS Unit. 13Data analysis 14PRO-Cap-Seq libraries were sequenced to a depth of 22-30 million reads while PRO-Seq libraries were 15 sequenced to a depth of 30-60 million reads (both: single-end, 50 bp).
Q10. How many AMPure XP beads were used to remove primers and adapters?
If necessary, 13 additional purification with AMPure XP beads was performed to remove primers and adapters 14 (purification with 1x AMPure XP beads; the supernatant was discarded and the DNA bound to the beads 15 subsequently eluted) or to exclude DNA fragments of more than 1 kb (purification with 0.54 x AMPure 16 XP beads; the high molecular weight fragments bound to the beads and were discarded, while the library 17 enriched for smaller DNA fragments remained in the supernatant).
Q11. How was the SV40 promoter removed from the luciferase plasm?
For assays with the endogenous 14 promoter, the SV40 promoter was removed from the luciferase-enhancer plasmids by restriction digestion 15 with BglII and HindIII-HF (both NEB).
Q12. What is the reason why the luciferase assays showed low enhancer?
The 5 individual elements of the Fgf5 enhancer cluster showed very low enhancer activity in classical luciferase 6 assays, even when combined with the endogenous promoter.
Q13. How many cycles for amplification of the ChIP-Seq libraries?
The number of cycles for optimal 25 PCR amplification was estimated to be 9-14 in total as described above for the ChIP-Seq libraries.
Q14. How does the effect of PE on expression levels in Drosophila be relieved?
When 28 placing PE outside of the intron and upstream of the promoter, this attenuating effect should be relieved 29 and the resulting expression levels should be higher than in a WT cell line.
Q15. What was the protocol for a biotin RNA enrichment?
In addition to this, the authors included a buffer exchange with a P-30 column - as 7 described in the PRO-Seq protocol - before the very first biotin-enrichment with Streptavidin beads.
Q16. Who has granted bioRxiv a license to display the preprint in perpetuity?
32.CC-BY-NC-ND 4.0 International licenseavailable under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Q17. How did the authors determine whether H3K27ac accumulation at the E1 enhancer was?
loss of E1 affected H3K27ac deposition at the E2 enhancer (Fig 5C), 15 and the authors observed reduced H3K27ac levels at the E1 enhancer upon E2 deletion (although not significant, 16 p-value=0.06).