GSM7508086: KRAB+Pfam memory screen, unbound fraction, br2; Homo sapiens; OTHER
Genomic DNA was extracted with the QIAgen Blood Maxi Kit following the manufacturer's instructions with up to 1.25 × 108 cells per column. DNA was eluted in EB and not AE to avoid subsequence PCR inhibition. The domain sequences were amplified by PCR with primers containing Illumina adapters as extensions (Table S3). A test PCR was performed using 5 μg of genomic DNA in a 50 μL (half-size) reaction to verify if the PCR conditions would result in a visible band at the expected size for each sample. Then, 12 - 24x 100 μL reactions were set up on ice (in a clean PCR hood to avoid amplifying contaminating DNA), with the number of reactions depending on the amount of genomic DNA available in each experiment. 10 μg of genomic DNA, 0.5 μL of each 100 μM primer, and 50 μL of NEBnext 2x Master Mix (NEB) was used in each reaction. The thermocycling protocol was to preheat the thermocycler to 98°C, then add samples for 3 minutes at 98°C, then 32x cycles of 98°C for 10 s, 63°C for 30 s, 72°C for 30 s, and then a final step of 72°C for 2 minutes. All subsequent steps were performed outside the PCR hood. The PCR reactions were pooled and ≥ 140 μL were run on at least three lanes of a 2% TBE gel alongside a 100-bp ladder for at least one hour, the library band around 395 bp was cut out, and DNA was purified using the QIAquick Gel Extraction kit (QIAgen) with a 30 ul elution into non-stick tubes (Ambion). A confirmatory gel was run to verify that small products were removed. These libraries were then quantified with a Qubit HS kit (Thermo Fisher), pooled with 15% PhiX control (Illumina), and sequenced on an Illumina NextSeq with a High output kit using a single end forward read (266 or 300 cycles) and 8 cycle index reads. From Tycko et al 2020: We queried the UniProt database (UniProt Consortium, 2015) for human genes that can localize to the nucleus. Subcellular location information on UniProt is determined from publications or 'by similarity' in cases where there is only a publication on a similar gene (e.g., ortholog) and is manually reviewed. We then retrieved Pfam-annotated domains using the ProDy searchPfam function (Bakan et al., 2011). We filtered for domains that were 80 amino acids or shorter and excluded the C2H2 Zinc finger DNA-binding domains, which are highly abundant, repetitive, and not expected to function as transcriptional effectors. We retrieved the sequence of the annotated domain and extended it equally on either side to reach 80 amino acids total. Duplicate sequences were removed, then codon optimization was performed for human codon usage, removing BsmBI sites and constraining GC content to between 20% and 75% in every 50 nucleotide window (performed with DNA chisel (Zulkower and Rosser, 2020)). 499 random controls of 80 amino acids lacking stop codons were computationally generated as controls. 362 elements tiling the DMD protein in 80 amino acid tiles with a 10 amino acid sliding window were also included as controls because DMD was not thought to be a transcriptional regulator. In total, the library consists of 5,955 elements (Table S1). Amplicons of the genome-integrated library members were generated via genomic extraction and subsequent PCR, and were sequenced using an Illumina NextSeq.