DOI: 10.7272/q6057d5n
title: Supporting data for: Three-dimensional genome re-wiring in loci with Human Accelerated Regions

description: Human Accelerated Regions (HARs) are conserved genomic loci that evolved
 at an accelerated rate in the human lineage and may underlie
 human-specific traits. We generated HARs and chimpanzee accelerated
 regions with an automated pipeline and an alignment of 241 mammalian
 genomes. Combining deep-learning with chromatin capture experiments in
 human and chimpanzee neural progenitor cells, we discovered a significant
 enrichment of HARs in topologically associating domains (TADs) containing
 human-specific genomic variants that change three-dimensional (3D) genome
 organization. Differential gene expression between humans and chimpanzees
 at these loci suggests rewiring of regulatory interactions between HARs
 and neurodevelopmental genes. Thus, comparative genomics together with
 models of 3D genome folding revealed enhancer hijacking as an explanation
 for the rapid evolution of HARs.
description: Lentivirus-based massively parallel reporter assay
 (lentiMPRA) library design and
 synthesis Tiles of 270bp in length were generated from all
 312 zooHARs. Multiple tiles were generated with a sliding window of 20bp
 if the zooHAR was longer than 270bp. In total, 549 oligos were designed to
 cover all zooHARs. We also included 143 oligos centered on active
 chromatin marks as positive controls. This oligo pool was synthesized by
 Twist Bioscience. Primary cortical cell culture for
 lentiMPRA De-identified tissue samples were collected with
 consent in strict observance of legal and institutional ethical
 regulations. Protocols were approved by the Human Gamete, Embryo, and Stem
 Cell Research Committee (institutional review board) at the University of
 California, San Francisco. Gestational week 18 cortical tissue was
 dissociated into a single-cell suspension using papain (LK003150,
 Worthington Biochemical) and plated on 15cm dishes coated with
 poly-O-lysine, laminin, and fibronectin. DMEM culture medium (Gibco) with
 B27 (Gibco) and PennStrep (Gibco) was changed every 24
 hours. Construction and sequencing of plasmid libraries
 for lentiMPRA LentiMPRA was performed as previously described
 with all modifications noted here. A 31bp minimal promoter and a 15bp
 random barcode were added to each lentiMPRA oligo through two rounds of
 PCR. The amplicon was then cloned into empty reporter backbone pLS-SceI
 (plasmid #137725, Addgene). Recombination products were amplified in
 electrocompetent cells (C3020, NEB) and grown in LB Agar plates
 (100217-214, VWR) at 37℃ overnight. We harvested ~3.5M colonies by
 Midiprep (12945, Qiagen), which yielded around 70 barcodes per oligo. A
 477bp region in the plasmid containing the oligo and barcode was amplified
 and sequenced in one lane of Illumina Nextseq Mid-Output to identify the
 barcodes associated with each oligo.
 Lentivirus packaging and
 infection Lentivirus production was performed following the
 manufacturer’s protocol (LT002, Genecopoeia). To achieve high titer, the
 crude solution was concentrated using Lenti-X concentrator (631232, Takara
 Bio). The concentrated virus was immediately stored at -80 in single-use
 aliquots. For each replicate, a cell counter was used to estimate cell
 density, and then 20 million primary cortical cells were plated and
 cultured in a 10cm dish for 2 days before infection. Each dish was
 infected with 500 ul of lentivirus to achieve a multiplicity of infection
 (MOI) of 85. Each barcode was estimated to be integrated into random loci
 over 200 times. Medium was refreshed the next day and cells were incubated
 for 2 days before harvesting DNA and RNA.
 DNA/RNA harvest and
 sequencing DNA and RNA were simultaneously extracted from
 infected samples using the Allprep kit (80204, Qiagen). To prepare
 sequencing libraries, 8μg RNA was reverse transcribed to generate cDNA
 using Superscript IV RT (Invitrogen; 18090200). The integrated barcodes in
 cDNA and 15μg gDNA were PCR amplified to add a unique molecular identifier
 (UMI), an index and Illumina P5/P7 sequence. DNA and RNA barcode libraries
 were pooled with a 1:3 molar ratio and sequenced with NextSeq
 High-Output. LentiMPRA computational
 analyses Sequencing libraries were batch corrected to
 account for differences between samples from different donors. Oligos were
 required to have at least 10 unique barcodes and exact match to the
 designed sequence. UMI-normalized reads per oligo were summed over all
 barcodes, and oligos with less than 40 total DNA reads were discarded. Out
 of 312 zooHARs, 276 passed these quality control steps. For each of these
 zooHARs, depth normalization was performed using counts per million reads
 sequenced (CPM), and a RNA CPM / DNA CPM ratio was calculated for each
 oligo in each replicate. A zooHAR was determined to be active if its
 maximally active tile had an average (over replicates) normalized RNA CPM
 / DNA CPM value exceeding the median value of this statistic for a set of
 positive control sequences with enhancer-associated epigenetic marks in
 neurodevelopment (median = 1.06). To compare machine learning predictions
 to lentiMPRA measurements, the 276 zooHARs passing lentiMPRA quality
 control were evaluated for whether they had activity above 1.06 (139
 active zooHARs) and/or a machine learning scores &gt; 0.3 (175
 predicted zooHARs), resulting in 88 high-confidence zooHAR
 neurodevelopmental enhancers validated by both
 approaches.