GSM7874181: Tissue culture, SPko-2-coIP, Synpo-coIP, biol rep 2; Mus musculus; RIP-Seq
Entorhino-hippocampal tissue cultures were prepared at postnatal day 3-5 from C57BL/6J (wildtype, Synpo+/+) and B6.129-Synpotm1Mndl/Dllr [Synpo-/-, (Deller et al., 2003; Lenz et al., 2021a; Lenz et al., 2021b)] mice of either sex as previously described (Del Turco and Deller, 2007). Synpo-/- strain was back crossed for at least 10 times to establish C57BL/6J genetic background. Cultivation medium contained 50% (v/v) MEM, 25% (v/v) basal medium eagle, 25% (v/v) heat-inactivated normal horse serum, 25 mM HEPES buffer solution, 0.15% (w/v) bicarbonate, 0.65% (w/v) glucose, 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamax. The pH was adjusted to 7.3. All tissue cultures were allowed to mature for at least 18 days in a humidified atmosphere with 5% CO2 at 35°C. Cultivation medium was replaced 3 times per week. Synpo-coIP: Six tissue cultures per biological replicate were washed in coIP-solution and dounce homogenized on ice in a solution containing (150 µl per sample, coIP-solution, in mM): 10 HEPES, 120 NaCl, 0.05% (v/v) NP-40 or IGEPAL CA-630 (Sigma Aldrich, #I8896), cOmplete protease inhibitor (Roche, according to manufacturer's instructions), murine RNase inhibitor (1:1000, New England Biolabs, #M0314) and cycloheximide (0.5 mg/ml, Sigma Aldrich, #C-4859). Input was precleared twice with protein A beads (10 µl per 100 µl sample/supernatant) to reduce non-specific binding. Protein A beads (10 µl, New England Biolabs, #S1425) were washed twice in coIP-solution and consecutively incubated for 1 hour at 4°C with an anti-synaptopodin antibody (rabbit anti-Synaptopodin, Synaptic Systems, #163002). Beads were carefully washed twice to remove unbound antibodies and incubated for 1 hour with the precleared supernatant ('input') at 4 °C with head-over-tail rotation. 10 µl of the precleared supernatant were kept as input control. After incubation, beads were washed twice with coIP-solution and finally dissolved in RNA protection buffer (New England Biolabs). RNA was released from the beads through protein K lysis (Monarch® Total RNA Miniprep Kit; New England Biolabs, #T2010S). After lysis, beads were removed from the solution in a magnetic rack and the RNA containing supernatant was transferred to fresh tubes. RNA isolation on coIP- and input-samples was then performed according to the manufacturer's instructions. RNA content was determined using the Agilent RNA 6000 Pico Kit (Agilent, #5067-1513) with a 2100 Bioanalyzer (Agilent, #G2939BA). RNA Library preparations for Synpo-related transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 Sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 150 bp read length. Regional mRNA library preparation: RNA Library preparations for transcriptome analysis were performed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (New England Biolabs, #E6420) according to the manufacturer's instructions. Briefly, isolation of the dentate gyrus and CA3 area from individual tissue cultures was performed using a scalpel. One isolated dentate gyrus or CA3 area respectively was transferred to 7.5 µl lysis buffer (supplemented with murine RNase inhibitor) and homogenized using a pestle. Samples were centrifuged for 30 s at 10,000 g and 5 µl of supernatant were collected from individual samples and further processed. After cDNA synthesis, cDNA amplification was performed according to the manufacturer's protocol with 12 PCR cycles. cDNA yield was consecutively analyzed by a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). cDNA amount was adjusted to 10 ng for further downstream applications. After fragmentation and adaptor ligation, dual index primers (New England Biolabs, #E7600S) were ligated in a library amplification step using 10 PCR cycles. Libraries were finally cleaned up with 0.8X SPRI beads following a standard bead purification protocol. Library purity and size distribution was assessed with a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 Sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 100 bp read length.