alias: GSM1563737
title: GSM1563737: B6WT-129KO control1 (SNP); Mus musculus; RNA-Seq
library_construction_protocol: After dissection brains were instantly weighed and homogenized in 10 volumes of Polysome Lysis Buffer (PLB) (Keene et al., 2006) containing 1 mM DTT, 400 µM vanadylribonucleoside complexes solution, 1x protease inhibitor cocktail (both Sigma) and 100 U/ml RNase inhibitor (Thermo Fisher Scientific) with 10 strokes of a dounce homogenizer at 800 rpm. Homogenates were subsequently centrifuged for 15 min at 16,000 g and 4°C. For each immunoprecipitation, 200 µl of the supernatant (input) was added to 760 µl of PLB and incubated with 1 µl DNase (New England Biolabs) on ice for 15 min. After the treatment, 40 µl 0.5 M EDTA, pH 8.0 (20 mM total) and 5 µl Triton-X-100 (0.5 % total) were added to each sample. Two preclearing steps of the lysate were performed by adding 0.9 mg of washed (PBST) and equilibrated (PLB) Dynabeads® Protein G (Life Technologies) and rotation for 30 min at 4°C. Double precleared lysates were incubated with 5 µg of a mouse isotype control antibody (Biolegend) for 45 min at 4°C before unspecific immunoprecipitates were removed by addition of 1.2 mg Dynabeads® Protein G (45 min, 4°C). PrP immunoprecipitation was done by incubation with 2 µg of SAF61 antibody (Cayman Chemical) for 1 h, 4°C and addition of 1.5 mg Dynabeads® Protein G (1 h, 4°C). The beads were then washed with 0.5 ml PLB without supplements for 5 times. For RNA cleanup, washed beads were resuspended in 200 µl PLB containing 0.1 % SDS and 0.4 µg/ml Proteinase K and incubated on a shaker at 700 rpm for 30 min and 50°C. RNA was extracted using phenol:chloroform:isoamylacohol (25:24:1), chloroform:isoamylacohol (24:1) and precipitated by adding 20 µl 3 M NaOAc pH 5.2, 1 µl glycogen (20 mg/ml) and 500 µl EtOH abs. Samples were incubated at -20°C over night and centrifuged in a precooled (4°C) tabletop centrifuge at maximum speed for 1 h. RNA pellet was washed with 500 µl 70 % EtOH, air-dried and resolved in 10 µl H20. RNA libraries were prepared according to Illumina's instructions, barcoded (Sample 1 - ACAGTG; Sample 2 - GCCAAT; Sample 3 - GTGAAA; Sample 4 - GTGGCC; Sample 5 - ATCACG; Sample 6 - CGATGT; Sample 7 - TTAGGC; Sample 8 - TGACCA), multiplexed (Sample 1,2,5-8 and 3-4, respectively) and run on an Illumina HiSeq2000™ (50 bp single end sequencing)
identifiers.namespace: GEO
identifiers.id: GSM1563737
attributes.tag: GEO Accession
attributes.value: GSM1563737