GSM9062043: Rett patient 4516 MECP2 mutant neurons; Homo sapiens; RNA-Seq
Brain tissue was cut on ice using a scalpel and homogenized in a glass dounce. The homogenate was then filtered through a 40-um cell strainer and centrifuged at 1000g for 8 min at 4ºC. The pellet was resuspended in a homogenization buffer and mixed with equal amounts of iodixanol. This mixture was then gently placed in a new tube over 29% iodixanol. The nuclei were centrifuged at 13500g for 20 min at 4ºC. The pellet was resuspended in the immunostaining buffer and incubated for 15 min at 4ºC. Next, primary antibody was added to the nuclei pellet and incubated on a rotator at 4ºC for 40 min. The following primary antibodies were used: rabbit MECP2 (Diagenode C15410052, 1:250), chicken NeuN (Millipore Sigma MAB377 1:250). Nuclei were centrifuged at 400 g for 5 min at 4ºC and washed with PBS/BSA twice. Secondary antibodies conjugated with Alexa 488 and 594 (1:500, Life Technologies A-21203, A21202) were added, accompanied by DAPI (Invitrogen 1:500), the nuclei were incubated on a rotator at 4ºC for 30 min. Immunostained nuclei were subjected to FACS. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. TruSeq Read 1 (read 1 primer sequence) is added to the molecules during GEM incubation. P5, P7, a sample index, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification.