title: GSM8879274: iN_day7_WT_SAR_seq; Homo sapiens; OTHER
library_construction_protocol: For SAR-seq, day7 iNs were incubated with 20 μM EdU for 18 hr. Cells were harvested and washed with PBS and incubated with accutase for 5-10 minutes. Cells were then collected using a cell scrapper (Corning), pelleted at 200xg for 5 minutes and resuspended in cold 0.1% BSA in PBS. Cold methanol was then added dropwise into all samples during slow vortexing to 80% final concentration. Cells were permeabilized on ice for 10 min and resuspended in 1 mL of Click-iT reaction solution for 2 hrs shaking at room temperature. Cells were then lysed with Proteinase K overnight at 37C. DNA was extracted with UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Invitrogen) according to manufacturer's instructions For SAR-seq, DNA was sonicated to 150-200 bp fragments using a Covaris S220 sonicator. Biotin-EdU labeled DNA fragments were pulled down using MyOne Streptavidin C1 Beads (ThermoFisher, cat. No. 650-01) and incubated at 24C shaking in a ThermoMixer C at 800 rpm for 30 min. Dynabeads were washed 3x in W&B (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 M NaCl, 0.1% Tween (freshly added)), 2x in EB (10 mM Tris-HCl pH 8.0), and 1x T4 DNA Ligase Buffer (NEB). Dynabeads were resuspended in end-repair reaction and incubated at 24C shaking at 800 rpm for 30 min. Dynabeads were washed 1x in 1X W&B, 2x EB, and 1x NEBuffer 2 (NEB) and resuspended in A-tailing reaction mix (NEB), followed by incubation at 37C shaking at 800 rpm for 30 min. Dynabeads were washed 1x in NEBuffer 2 and resuspended in ligation reaction mix (NEB), 5 nM annealed TruSeq truncated adapter and incubated at 25C shaking at 600 rpm for 20 min. Ligation reaction was stopped by adding 50 mM EDTA, and Dynabeads were washed 3x in 1X W&B, 3x EB. PCR amplified for 16 cycles with Kapa HiFI HotStart Ready Mix using TruSeq index adapters. PCR products were separated from DynaBeads and cleaned using AMPure Beads XP.
identifiers.namespace: GEO
identifiers.id: GSM8879274_r1