title: GSM5100425: sgControl SAR-seq for sgPOLB in iNeuron rep2; Homo sapiens; OTHER
library_construction_protocol: Cells were washed with PBS, incubated with accutase for 5-10 mins, collected with a cell scrapper, pelleted at 200 × g for 5 minutes and resuspended in cold 0.1% BSA in PBS. Cold methanol was then added dropwise during slow vortexing to 80% final concentration. Samples were kept on ice for 20 minutes and then stored at -20C until processing. Copper catalyzes azide-alkyne click chemistry. For biotin labeling via Click-iT reaction, cells were first washed 1x in PBS, permeabilized with 0.2% Triton-X100/PBS for 10 minutes on ice, and then washed 1x in PBS. Then the following were added in order: 3 mM copper sulfate (Sigma), 50 M biotin azide (ThermoFisher, Cat. No. B10184), and 1X Click-iT additive (ThermoFisher, Cat. No. C10424) for 2 hours shaking at room temperature. Cells were then washed one time in PBS and lysed in 50 mM Tris pH 8.0 with 1% SDS and Proteinase K overnight at 37°C. DNA was extracted with UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Invitrogen) according to manufacturer's instructions, followed by 2.5:1 volume ethanol and 1:10 volume sodium acetate precipitation. DNA pellets were resuspended in TE buffer and sheared to 150-200 bp fragments using Covaris S220 sonicator at 10% duty cycle, 175 peak incident power, 200 cycles per burst, for 240 seconds. DNA was again precipitated by 2.5:1 volume ethanol and 1:10 volume sodium acetate and resuspended in TE buffer. Biotin-EdU fragments were pulled down using MyOne Streptavidin C1 Beads (ThermoFisher, Cat. No. 650-01). Before pulldown, 35 L of Dynabeads were washed two times with 1 mL 1X Wash and Binding buffer (1X W&B) (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 M NaCl, 0.1% Tween20) on a DynaMag-2 magnetic separator (Invitrogen, Cat. No. 12321D) and resuspended in 2X W&B (10 mM Tris-HCl pH8.0, 2 mM EDTA, 2 M NaCl, 0.2% Tween20). Equal volume of Dynabeads in 2X W&B were added to DNA in TE and incubated at 24°C shaking in a ThermoMixer C at 800 rpm for 30 minutes. Dynabeads bound to biotin-EdU fragments were washed three times in 1 mL of 1X W&B, two times in 1 mL EB, and one time in 1mL 1X T4 DNA Ligase Buffer (NEB). Dynabeads were resuspended in 50 L end-repair reaction mix (1X T4 DNA Ligase Buffer, 0.4 mM of dNTPs, 2.7 U of T4 DNA polymerase (NEB), 9 U of T4 Polynucleotide Kinase (NEB), and 1 U of Klenow fragment (NEB)) and incubated at 24°C shaking at 800 rpm for 30 minutes. Dynabeads were washed one time in 1mL 1X W&B, two times in 1mL EB, and one time in 1 mL NEBuffer 2 (NEB) and resuspended in 50 L of A-tailing reaction mix (1X NEB dA-Tailing Buffer and 20 U Klenow fragment exo- (NEB)), followed by incubation at 37°C shaking at 800 rpm for 30 minutes. Dynabeads were then washed again 1x in 1 mL NEBuffer 2 and resuspended in 115 L of ligation reaction mix (1X Quick Ligase Buffer (NEB), 6000 U Quick Ligase (NEB), 5 nM annealed TruSeq truncated adapter) and incubated at 25°C shaking at 600 rpm for 20 minutes. Ligation reaction was stopped by adding 50 mM EDTA, and Dynabeads were washed three times in 1 mL 1X W&B, three times in 1 mL EB, and finally resuspended in 8 L EB + 10 L 2X Kapa HiFI HotStart Ready Mix (Kapa Biosciences). 10 mM primers 5'-CAAGCAGAAGACGGCATACGA- GATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T-3' and 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCC CTACACGACGCTCTTCCGATC*T-3' (* indicates a phosphothiorate bond and a NNNNNN TruSeq index sequence) were added with 37 L PCR reaction mix (20 L 2X Kapa HiFI HotStart Ready Mix, 17 L H2O) for final volume of 60 L. DNA was amplified using PCR program: 98°C, 45 seconds; 15 cycles [98°C, 15 seconds; 63°C, 30 seconds; 72°C, 30 seconds]; 72°C, 5 minutes. PCR products were separated from DynaBeads and cleaned using 1.8X volume AMPure Beads XP. 150-200 bp bands were isolated on 2% agarose gel and purified using QIA-quick Gel Extraction Kit (Qiagen). Prior to sequencing using Illumina NextSeq 550 (75 bp single read), library concentrations were calculated by KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems).    To specifically sequence only EdU-incorporated strands of DNA (Strand-Specific SAR-seq), SAR-seq was followed exactly as above with additional steps prior to PCR amplification. After washing three times in 1 mL 1X W&B and three times in 1 mL EB post-ligation, Dynabeads were washed in 50 L 1X SSC buffer and resuspended in 20 L of 0.15 M NaOH for 10 minutes at room temperature to denature DNA strands. Beads were placed back on the DynaMag-2 magnetic separator and washed one time with 20 L 0.1 M NaOH, one time with 1 mL 1X W&B, two times with 1 mL EB, and resuspended in 8 L EB + 10 L 2X Kapa HiFI HotStart Ready Mix. Primers and PCR reaction mix were added as above.