title: GSM5105578: SIM_PlpCreRibo_Mu_T3_1_IgG; Mus musculus; RNA-Seq
library_construction_protocol: Samples were immunoprecipitated at the Weizman Instiute (Israel) according to the published protocol4, 5. In brief, samples were homogenized in 4 mL cold homogenization buffer (50 mM Tris, pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:100 protease inhibitor (Sigma), 200 U/mL RNasin (Promega) and 0.1 mg/ml cycloheximide (Sigma)) with a Dounce homogenizer (Sigma) until the suspension was homogeneous. To remove cell debris, 2 mL of the homogenate was transferred to a microcentrifuge tube and centrifuged at 10,000 g at 4°C for 10 min. Supernatants were transferred and split, and 8 μL (10 µg) of anti-HA antibody (H9658, Sigma) or 20 μL (10 µg) of mouse monoclonal IgG1 antibody (Sigma, Cat# M5284) was added to the supernatant, followed by 4h of incubation with slow rotation at 4°C. Meanwhile, Dynabeads Protein G (Thermo Fisher Scientific), were equilibrated to homogenization buffer by washing three times. At the end of 4 h of incubation with antibody, beads were added to each sample, followed by incubation overnight at 4°C. Next, samples were washed three times with high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:200 protease inhibitor, 100 U/ml RNasin and 0.1 mg/ml cycloheximide). At the end of the washes, beads were magnetized and excess buffer was removed, 150 µl lysis buffer was added to the beads and RNA was extracted with Dynabeads mRNA Direct purification kit (Thermo Fisher) according to manufacturer's instructions. RNA was eluted in 6 μl H2O and used for RNA-seq mRNA was captured with Dynabeads oligo(dT) (Life Technologies) according to manufacturer's guidelines. A bulk variation of MARSseq6 was used to prepare libraries for RNA-seq. RNA was reversed transcribed with MARSseq barcoded RT primer in a 10 mL volume with the Affinity Script kit (Agilent). Reverse transcription was analyzed by qRT-PCR and samples with a similar CT were pooled (up to eight samples per pool). Each pool was treated with Exonuclease I (NEB) for 30 min at 37°C and subsequently cleaned by 1.2 x volumes of SPRI beads (Beckman Coulter). Subsequently, the cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20mL reaction, incubating for 2 h at 16°C. The product was purified with 1.4 x volumes of SPRI beads, eluted in 8 mL and in vitro transcribed (with the beads) at 37°C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37°C and the amplified RNA (aRNA) purified with 1.2 volumes of SPRI beads. The aRNA was fragmented by incubating 3 min at 70°C in Zn2+ RNA fragmentation reagents (Ambion) and purified with 2 x volumes of SPRI beads. The aRNA was ligated to the MARS-seq ligation adaptor with T4 RNA Ligase I (NEB). The reaction was incubated at 22°C for 2 h. After 1.5 x SPRI cleanup, the ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent) and a primer complementary to the ligated adaptor. The reaction was incubated for 2 min at 42°C, 45 min at 50°C, and 5 min at 85°C. The cDNA was purified with 1.5 x volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5mM of P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kappa Biosystems). The amplified pooled library was purified with 0.7 x volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument