Q2. How many cycles of PCR were added to the illumina library?
PE2 indexing barcodes were then 528 added by amplifying 2 microliters of the previous reaction with forward primer P1_outer and 529 reverse primers PE2_outer_SIC69 and PE2_outer_SIC70 (Supplementary file 6) for 5 cycles 530 at an annealing temperature of 66C followed by 5 cycles with no annealing step (NEB Q5) and 531 then purified with the Monarch PCR kit.
Q3. How many barcodes were cloned into oligos?
To clone the basal promoter 475 into barcoded oligos without any upstream cis-regulatory sequence, the authors placed the SpeI site 47623next to the EcoRI site, which allowed us to place the promoter between the EcoRI site and the 477 3’ barcode.
Q4. How many reads were filtered to ensure that the barcode sequence perfectly matched the expected?
Sequencing reads were filtered to ensure that the barcode sequence perfectly matched the 541 expected sequence (>93% reads in a sample for the Rho libraries, >86% reads for the 542 Polylinker libraries).
Q5. What is the significance of the CRX motifs in the ATAC-seq?
After synthesis of their library, the authors discovered 434 11% of these sequences do not actually overlap H3K27ac ChIP-seq peaks (110/1004 of the 435 H3K4me3- group and 60/541 of the H3K4me3+ group), but the authors still included them in the analysis 436 because they contain CRX motifs in ATAC-seq peaks.
Q6. What was the log-normal distribution of expression levels for each sequence?
Expression levels were approximately log-normally distributed, so the authors computed 566 the log-normal parameters for each sequence and then performed Welch’s t-test.
Q7. what is the cis-regulatory 956 logic of hedgehog gradient?
The cis-regulatory 956 logic of Hedgehog gradient responses: key roles for gli binding affinity, competition, and 957 cooperativity.
Q8. what is the probability of finding a spurious motif in a mammalian genome?
For TFs and a 681bp window (which is the size of their sequences), and , meaning 682 that five total motifs for three different TFs specifies an approximately unique 164 bp location in 683 a mammalian genome.
Q9. What was the basal promoter construct in the HOCOMOCO database?
There was no basal promoter construct in this 715 library, so instead the authors defined silencers as ChIP-seq peaks that were at least two-fold below the 716 log2 mean of all scrambled sequences.
Q10. What was the common method used to determine which genes are near a member of their library?
The authors 591 determined which differentially expressed genes are near a member of their library using 592 previously published associations between retinal ATAC-seq peaks and genes [REF Murphy 593 2019].
Q11. What is the way to use the Python 692 wrappers?
The authors wrote custom Python 692 wrappers for gkmSVM to allow for interfacing between the C++ binaries and the rest of their 693 workflow.
Q12. How many motifs can be found in a genome of length?
Five total motifs for three different TFs can be achieved in two ways: 684 three motifs for A, one for B, and one for C (3-1-1), or two motifs for A, two for B, and one for C 685(2-2-1).
Q13. what is the cis-regulatory function of 1029 enhancer?
Massively parallel in vivo 1029 enhancer assay reveals that highly local features determine the cis-regulatory function of 1030 ChIP-seq peaks.
Q14. What is the entropy of a cis-regulatory sequence?
Because each arrangement is equally likely, 652then is also the expected value of and the authors can write the entropy as 653, which is Shannon entropy.