GSM7429110: AT 7 RNAseq cerebellum; Homo sapiens; RNA-Seq
PAR ChIP: The protocol was adapted from (16). Briefly, SH-SY5Y cells were differentiated as described above and two 15 cm dishes were used for each ChIP replicate (approximately 20M cells). Cells were removed from dishes using trypsin and then resuspended in media in a volume of 36 ml. Formaldehyde (37%, Sigma F1635) was added to 1% final concentration and incubated at 37°C for 7 min. with occasional inversion of the tube. 2.5M glycine was added to a final concentration of 0.125M. The tube was inverted several times and cells recovered by centrifugation at 550xg for 5 min. at 4°C. The pellet was washed twice with 10 ml cold PBS using 550xg centrifugation for 5 min. each time. The pellets were snap frozen using liquid nitrogen and stored at -80°C for later use. Each cell pellet was resuspended in 1mL cold PAR ChIP RIPA buffer (10 mM Tris-HCl, pH7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100) containing protease inhibitor (Fisher, A32955). Samples were sonicated using a Diagenode Bioruptor UCD-200 instrument on high power with 15 sec on/ 15 sec off cycles for 45 min. at 4°C. Under these conditions, fragmented chromatin was 200 to 500 bp. Samples were centrifuged at 20,000xg for 10 minutes at 4°C. The supernatant was transferred to another tube; 50 μl were reserved as input DNA. 40 μl of Protein A/G magnetic beads was incubated with 5 μg anti-pan-ADP-ribose binding reagent (Millipore Sigma, MABE1016) per IP sample in 100 μl PBS for 30 min. then washed with 200μl PBS twice. The beads were then added to the supernatant and incubated at 4°C overnight. The following day, beads were washes for 10 min. at 4°C with the following sequence of buffers: 2x with 1 mL of RIPA buffer, 2x with 1 mL of RIPA buffer + 0.3M NaCl, 1x with 1 mL of TE pH 8 + 0.2% Triton X-100, 1x with 1 mL of TE pH 8. The beads were resuspended in 100μl TE; 3μl 10% SDS and 5μl 20 mg/ml proteinase K were added and the samples incubated at 65°C for 4 hrs while rocking. The tubes were vortexed every 30 to 50 min. The DNA was removed from beads using a magnetic stand; 100μl TE + 0.5M NaCl was added to the beads and then removed and combined with the first sample. 200 μl phenol/chloroform/isoamyl alcohol was added and samples were added to Phase Lock gel columns (VWR, 10847-802) and processed according to manufacturer instructions. Samples were then ethanol precipitated by adding 2μl glycogen (20 mg/ml), 20μl 3M NaOAc pH 5.2, and 500 μl cold ethanol (100%). After incubation on dry ice for 15 min., samples were centrifuged at 20,000xg for 15 min. at 4°C. Pellets were washed with 70% ethanol twice; the supernatant was removed and pellets dried at room temp. Pellets were resuspended in 22 μl 10 mM Tris-HCl pH 8.0, vortexed, and stored at -20°C.  R-ChIP  The protocol was adapted from (24). Briefly, SH-SY5Y cells were differentiated as described above and two 15 cm dishes were used for each ChIP replicate (approximately 20M cells). Cells were crosslinked in the dishes with the addition of formaldehyde to 1% final concentration and incubated for 7 min. while shaking (120 RPM). 2.5M glycine was added to a final concentration of 0.125M. Cells were removed by scraping and transferred to conical tubes with an additional 5 ml PBS to wash the dishes. Cells were centrifuged at 2187xg for 15 min. at 4°C, then washed with cold PBS and pelleted again. The pellets were snap frozen using liquid nitrogen and stored at -80°C for later use. Each cell pellet was resuspended in 2.3 mL cold R-ChIP RIPA buffer (50 mM Tris-HCl, pH 8.0, 0.15M NaCl, 2 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 0.1% Igepal CA-630 (NP40) containing protease inhibitor (Fisher, A32955) and incubated on ice for 10 min. Samples were sonicated using a Diagenode Bioruptor UCD-200 instrument on high power with 15 sec on/ 15 sec off cycles for 45 min. at 4°C. Samples were centrifuged at 3,889xg for 10 minutes at 4°C. The supernatant was transferred to another tube; 50 μl were reserved as input DNA. 25 μl of Protein A/G magnetic beads per IP sample were washed with 1 ml wash buffer (20 mM Tris-HCl pH 8.0, 0.15M NaCl, 2 mM EDTA, 1% Triton X-100) using a magnetic separator; this step was repeated for a total of 3 washes. Beads were resuspended in bead blocking buffer (0.2 μg/ml BSA, 0.2 mg/ml glycogen in PBS) and incubated for 1 hr at RT. Beads were then washed 3 times with antibody binding buffer (5 μg/ml BSA in PBS), resuspended in 250 μl antibody binding buffer and 2.5 μg FLAG M2 antibody (Sigma) per 25 μl of Protein A/G magnetic beads and incubated overnight at 4°C with rotation. After removal of unbound antibody, beads were resuspended in 1 ml sonicated lysate and incubated overnight at 4°C with rotation. The following day, beads were washed 3 times for 3 min. at room temperature with 1 ml wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) containing protease inhibitor, then 3 times with wash buffer II (wash buffer I containing 0.5M NaCl), then once with wash buffer III (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP40, 0.25M LiCl), then once with TE. Samples were moved to a new tube and eluted with 100 μl elution buffer (0.1 M sodium bicarbonate, 1% SDS) at 30 °C for 30 min. Beads were removed, 1 μl RNase A (20 mg/ml) was added to each sample and supernatants were decrosslinked at 65°C for 22 hrs. 1 μl Proteinase K (20 mg/ml) was added to each sample and incubated for an additional 2 hrs at 65°C. Samples were purified using the nucleotide removal kit (Qiagen) according to manufacturer instructions. RNA extraction from SH-SY5Y cells: Total RNA extraction was performed using NEB Monarch Total RNA Miniprep Kit (New England Biolabs Cat #T2010S). RNAseq library prep was performed using NEBNext Ultra II RNA Library Prep for Illumina (NEB Cat# E7770S). The NEB protocol was followed using the Poly(A) mRNA Magnetic Isolation Module and SPRIselect beads (Beckman Coulter Cat #B23317). Library adapter was diluted 5-fold. The additional SPRIselect cleanup step referred to at 1.11.3 in the NEB protocol was followed.RNA extraction from patient tissues: Fresh-frozen A-T patient and control tissues were obtained from the NIH Neurobiobank (Table S1). 50mg patient tissue RNA was extracted in an RNase free environment using RNA Extraction kit for Bioruptor Plus (Diagenode Cat# C20000010). The additional protocol listed for DNaseI treatment was followed for 10ug of extracted RNA per sample. DNaseI treated RNA was cleaned using RNeasy Mini Kit (Qiagen Cat# 74104). ChIP library preparation and sequencing  For PAR and R-ChIP, the eluted DNA (2 biological replicates per condition) as well as input samples were used to make sequencing libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) with NEBNext Multiplex dual index primers using 12 amplification cycles and 2 additional AMPure XP clean-up steps at 0.8X. Libraries were sequenced by the UT Genomic Sequencing and Analysis Facility (RRID:SCR_021713) using the Illumina NovaSeq SP platform with PE150 runs. RNAseq library prep was performed using NEBNext Ultra II RNA Library Prep for Illumina (NEB Cat# E7770S). The NEB protocol was followed using the Poly(A) mRNA Magnetic Isolation Module and SPRIselect beads (Beckman Coulter Cat #B23317). Library adapter was diluted 5-fold. The additional SPRIselect cleanup step referred to at 1.11.3 in the NEB protocol was followed.