alias: GSM2464501
title: GSM2464501: IgG-1; Mus musculus; RIP-Seq
library_construction_protocol: Sorted LT-HSCs were lysed in hypotonic buffer containing 10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT to remove cytoplasmic contents. Nuclear pellets were lysed in RIPA lysis buffer for 1 h, followed by incubation with anti-cGAS antibody for 4 h post preclearance with protein A/G agarose. cGAS was then immunoprecipitated by protein A/G agarose. Immunoprecipitates were washed with RIPA wash buffer. RNA was extracted by resuspending the cGAS-RNA complex in TRIzol reagent according to manufacturer’s instructions The retrieved RNA is fragmented by adding First Strand Master Mix（Invitrogen）. First-strand cDNAis generated by First Strand Master Mix and Super Script II reverse transcription (Invitrogen) (Reaction condition：25℃for 10 min；42℃for 50 min；70℃for 15 min).Purify the product(Agencourt RNAClean XP Beads，AGENCOURT), then add Second Strand Master Mix and dATP,dGTP, dCTP, dUTP mix to synthesize the second-strand cDNA (16℃for 1h).Then the purified cDNA combine with End Repair Mix, incubate at 30℃for 30min. After purification with beads, add A-Tailing Mix , incubate at 37℃for 30 min. Combine the Adenylate 3'Ends DNA, Index Adapter and Ligation Mix, incubate at 30℃for 10 min. After that, add the Uracil-N-Glycosylase enzyme into the purified ligation product, incubate at 37℃for 10min. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the cDNA fragments. Then the PCR products are purified with Ampure XP Beads.
identifiers.namespace: GEO
identifiers.id: GSM2464501